Endothelial cells are reported to be glycolytic and to minimally rely on mitochondria for ATP generation. Rather than providing energy, mitochondria in endothelial cells may act as signaling organelles that control cytosolic Ca2+ signaling or modify reactive oxygen species (ROS). To control Ca2+ signaling, these organelles are often observed close to influx and release sites and may be tethered near Ca2+ transporters. In this study, we used high-resolution, wide-field fluorescence imaging to investigate the regulation of Ca2+ signaling by mitochondria in large numbers of endothelial cells (∼50 per field) in intact arteries from rats. We observed that mitochondria were mostly spherical or short-rod structures and were distributed widely throughout the cytoplasm. The density of these organelles did not increase near contact sites with smooth muscle cells. However, local inositol trisphosphate (IP3)-mediated Ca2+ signaling predominated near these contact sites and required polarized mitochondria. Of note, mitochondrial control of Ca2+ signals occurred even when mitochondria were far from Ca2+ release sites. Indeed, the endothelial mitochondria were mobile and moved throughout the cytoplasm. Mitochondrial control of Ca2+ signaling was mediated by ATP production, which, when reduced by mitochondrial depolarization or ATP synthase inhibition, eliminated local IP3-mediated Ca2+ release events. ROS buffering did not significantly alter local Ca2+ release events. These results highlight the importance of mitochondrial ATP production in providing long-range control of endothelial signaling via IP3-evoked local Ca2+ release in intact endothelium.

Blood flow, blood clotting, angiogenesis, vascular permeability, and vascular remodeling are each controlled by a large number of variable, noisy, and interacting chemical inputs to the vascular endothelium. The endothelium processes the entirety of the chemical composition to which the cardiovascular system is exposed, carrying out sophisticated computations that determine physiological output. Processing this enormous quantity of information is a major challenge facing the endothelium. We analyzed the responses of hundreds of endothelial cells to carbachol (CCh) and adenosine triphosphate (ATP) and found that the endothelium segregates the responses to these two distinct components of the chemical environment into separate streams of complementary information that are processed in parallel. Sensitivities to CCh and ATP mapped to different clusters of cells, and each agonist generated distinct signal patterns. The distinct signals were features of agonist activation rather than properties of the cells themselves. When there was more than one stimulus present, the cells communicated and combined inputs to generate new distinct signals that were nonlinear combinations of the inputs. Our results demonstrate that the endothelium is a structured, collaborative sensory network that simplifies the complex environment using separate cell clusters that are sensitive to distinct aspects of the overall biochemical environment and interactively compute signals from diverse but interrelated chemical inputs. These features enable the endothelium to selectively process separate signals and perform multiple computations in an environment that is noisy and variable.

Chronic inflammation predisposes to aging-associated disease, but it is unknown whether immunity regulation might be important for extending healthy lifespan. Here we show that in C. elegans, dietary restriction (DR) extends lifespan by modulating a conserved innate immunity pathway that is regulated by p38 signaling and the transcription factor ATF-7. Longevity from DR depends upon p38-ATF-7 immunity being intact but downregulated to a basal level. p38-ATF-7 immunity accelerates aging when hyperactive, influences lifespan independently of pathogen exposure, and is activated by nutrients independently of mTORC1, a major DR mediator. Longevity from reduced insulin/IGF-1 signaling (rIIS) also involves p38-ATF-7 downregulation, with signals from DAF-16/FOXO reducing food intake. We conclude that p38-ATF-7 is an immunometabolic pathway that senses bacterial and nutrient signals, that immunity modulation is critical for DR, and that DAF-16/FOXO couples appetite to growth regulation. These conserved mechanisms may influence aging in more complex organisms.

The apparently stationary phase of mitochondrial motion was investigated in epithelial cells by spinning disk confocal light microscopy combined with image correlation based single particle tracking using custom software producing sub-pixel accuracy measurements (approximately 5 nm) at 10-12 Hz frame-rates. The analysis of these data suggests that the previously described stationary, or anchored phase, in mitochondrial movement actually comprise Brownian diffusion, interspersed with frequent and brief motor-driven events whose duration are stochastically determined. We have therefore discovered a new aspect of mitochondrial behavior, which we call stochastically determined, directed movement.

Aging is the summation of many subtle changes which result in altered cardiovascular function. Impaired endothelial function underlies several of these changes and precipitates plaque development in larger arteries. The endothelium transduces chemical and mechanical signals into changes in the cytoplasmic calcium concentration to control vascular function. However, studying endothelial calcium signaling in larger arteries in a physiological configuration is challenging because of the requirement to focus through the artery wall. Here, pressure- and agonist-sensitive endothelial calcium signaling was studied in pressurized carotid arteries from young (3-month-old) and aged (18-month-old) rats by imaging from within the artery using gradient index fluorescence microendoscopy. Endothelial sensitivity to acetylcholine increased with age. The number of cells exhibiting oscillatory calcium signals and the frequency of oscillations were unchanged with age. However, the latency of calcium responses was significantly increased with age. Acetylcholine-evoked endothelial calcium signals were suppressed by increased intraluminal pressure. However, pressure-dependent inhibition of calcium signaling was substantially reduced with age. While each of these changes will increase endothelial calcium signaling with increasing age, decreases in endothelial pressure sensitivity may manifest as a loss of functionality and responsiveness in aging.

The adaptive optics scanning laser ophthalmoscope (AOSLO) was first developed in 2002 and since then the technology has been adopted in several laboratories around the world, for both clinical and psychophysical research. There have been a few major design implementations of the AOSLO. The first used on-axis tilted spherical mirrors in a planar arrangement, and the second minimized the build up of astigmatism present in the first design by using a non-planar arrangement. Other designs have avoided astigmatism by using custom-made toroidal mirrors or by using lenses on-axis, rather than mirrors. We present a new design implementation for an AOSLO that maintains a planar optical alignment without the build up astigmatism using compact, reconfigurable modules based on an Offner relay system. We additionally use an off-the-shelf digital oscilloscope for data capture and custom-written Python code for generating and analyzing the retinal images. This design results in a compact system that is simple to align and, being composed of modular relays, has the potential for additional components to be added. We show that this system maintains diffraction-limited image quality across the field of view and that cones are clearly resolved in the central retina. The modular relay design is generally applicable to any system requiring one or more components in the pupil conjugate plane. This is likely to be useful for any point-scanned system, such as a standard scanning laser ophthalmoscope or non-ophthalmic confocal imaging system.

The mitochondrial unfolded protein response (mitoUPR) is a stress response pathway activated by disruption of proteostasis in the mitochondria. This pathway has been proposed to influence lifespan, with studies suggesting that mitoUPR activation has complex effects on longevity.

Organ and tissue development are highly coordinated processes; lens growth and functional integration into the eye (emmetropia) is a robust example. An epithelial monolayer covers the anterior hemisphere of the lens, and its organization is the key to lens formation and its optical properties throughout all life stages. To better understand how the epithelium supports lens function, we have developed a novel whole tissue imaging system using conventional confocal light microscopy and a specialized analysis software to produce three-dimensional maps for the epithelium of intact mouse lenses. The open source software package geometrically determines the anterior pole position, the equatorial diameter, and three-dimensional coordinates for each detected cell in the epithelium. The user-friendly cell maps, which retain global lens geometry, allow us to document age-dependent changes in the C57/BL6J mouse lens cell distribution characteristics. We evidence changes in epithelial cell density and distribution in C57/BL6J mice during the establishment of emmetropia between postnatal weeks 4-6. These epithelial changes accompany a previously unknown spheroid to lentoid shape transition of the lens as detected by our analyses. When combined with key findings from previous mouse genetic and cell biological studies, we suggest a cytoskeleton-based mechanism likely underpins these observations.

Post-translational changes in the redox state of cysteine residues can rapidly and reversibly alter protein functions, thereby modulating biological processes. The nematode C. elegans is an ideal model organism for studying cysteine-mediated redox signaling at a network level. Here we present a comprehensive, quantitative, and site-specific profile of the intrinsic reactivity of the cysteinome in wild-type C. elegans. We also describe a global characterization of the C. elegans redoxome in which we measured changes in three major cysteine redox forms after H2O2 treatment. Our data revealed redox-sensitive events in translation, growth signaling, and stress response pathways, and identified redox-regulated cysteines that are important for signaling through the p38 MAP kinase (MAPK) pathway. Our in-depth proteomic dataset provides a molecular basis for understanding redox signaling in vivo, and will serve as a valuable and rich resource for the field of redox biology.

Age is proposed to be associated with altered structure and function of mitochondria; however, in fully-differentiated cells, determining the structure of more than a few mitochondria at a time is challenging. In the present study, the structures of the entire mitochondrial complements of cells were resolved from a pixel-by-pixel covariance analysis of fluctuations in potentiometric fluorophore intensity during 'flickers' of mitochondrial membrane potential. Mitochondria are larger in vascular myocytes from aged rats compared to those in younger adult rats. A subpopulation of mitochondria in myocytes from aged, but not younger, animals is highly-elongated. Some mitochondria in myocytes from younger, but not aged, animals are highly-motile. Mitochondria that are motile are located more peripherally in the cell than non-motile mitochondria.

Increased pressure suppresses endothelial control of vascular tone but it remains uncertain (1) how pressure is sensed by the endothelium and (2) how the vascular response is inhibited. This study used a novel imaging method to study large numbers of endothelial cells in arteries that were in a physiological configuration and held at normal blood pressures. Increased pressure suppressed endothelial IP3 -mediated Ca(2+) signals. Pressure modulated endothelial cell shape. The changes in cell shape may alter endothelial Ca(2+) signals by modulating the diffusive environment for Ca(2+) near IP3 receptors. Endothelial pressure-dependent mechanosensing may occur without a requirement for a conventional molecular mechanoreceptor.

Compounds with lifespan extension activity are rare, although increasing research efforts have been invested in this field to find ways to extend healthy lifespan. By applying a yeast-based high-throughput assay to identify the chronological lifespan extension activity of mulberry extracts rapidly, we demonstrated that a group of prenylated flavones, particularly morusin and mulberrin, could extend the chronological lifespan of budding yeast via a nutrient-dependent regime by at least partially targeting SCH9. Their antiaging activity could be extended to C. elegans by promoting its longevity, dependent on the full functions of genes akt-1 or akt-2. Moreover, additional benefits were observed from morusin- and mulberrin-treated worms, including increased reproduction without the influence of worm health (pumping rate, pumping decline, and reproduction span). In the human HeLa cell model, morusin and mulberrin inhibited the phosphorylation of p70S6K1, promoted autophagy, and slowed cell senescence. The molecular docking study showed that mulberrin and morusin bind to the same pocket of p70S6K1. Collectively, our findings open up a potential class of prenylated flavones performing their antiaging activity via nutrient-sensing pathways.

While mitochondrial function is essential for life in all multicellular organisms, a mild impairment of mitochondrial function can extend longevity in model organisms. By understanding the molecular mechanisms involved, these pathways might be targeted to promote healthy aging. In studying two long-lived mitochondrial mutants in C. elegans, we found that disrupting subunits of the mitochondrial electron transport chain results in upregulation of genes involved in innate immunity, which is driven by the mitochondrial unfolded protein response (mitoUPR) but also dependent on the canonical p38-mediated innate immune signaling pathway. Both of these pathways are required for the increased resistance to bacterial pathogens and extended longevity of the long-lived mitochondrial mutants, as is the FOXO transcription factor DAF-16. This work demonstrates that both the p38-mediated innate immune signaling pathway and the mitoUPR act in concert on the same innate immunity genes to promote pathogen resistance and longevity and that input from the mitochondria can extend longevity by signaling through these pathways. This indicates that multiple evolutionarily conserved genetic pathways controlling innate immunity also function to modulate lifespan.

Mitochondrial morphology is central to normal physiology and disease development. However, in many live cells and tissues, complex mitochondrial structures exist and morphology has been difficult to quantify. We have measured the shape of electrically-discrete mitochondria, imaging them individually to restore detail hidden in clusters and demarcate functional boundaries. Stochastic "flickers" of mitochondrial membrane potential were visualized with a rapidly-partitioning fluorophore and the pixel-by-pixel covariance of spatio-temporal fluorescence changes analyzed. This Flicker-assisted Localization Microscopy (FaLM) requires only an epifluorescence microscope and sensitive camera. In vascular myocytes, the apparent variation in mitochondrial size was partly explained by densely-packed small mitochondria. In normotensive animals, mitochondria were small spheres or rods. In hypertension, mitochondria were larger, occupied more of the cell volume and were more densely clustered. FaLM provides a convenient tool for increased discrimination of mitochondrial architecture and has revealed mitochondrial alterations that may contribute to hypertension.

Human lens regeneration and the Bag-in-the-Lens (BIL) surgical treatment for cataract both depend upon lens capsule closure for their success. Our studies suggest that the first three days after surgery are critical to their long-term outcomes. Using a rat model of lens regeneration, we evidenced lens epithelial cell (LEC) proliferation increased some 50 fold in the first day before rapidly declining to rates observed in the germinative zone of the contra-lateral, un-operated lens. Cell multi-layering at the lens equator occurred on days 1 and 2, but then reorganised into two discrete layers by day 3. E- and N-cadherin expression preceded cell polarity being re-established during the first week. Aquaporin 0 (AQP0) was first detected in the elongated cells at the lens equator at day 7. Cells at the capsulotomy site, however, behaved very differently expressing the epithelial mesenchymal transition (EMT) markers fibronectin and alpha-smooth muscle actin (SMA) from day 3 onwards. The physical interaction between the apical surfaces of the anterior and posterior LECs from day 3 after surgery preceded cell elongation. In the human BIL sample fibre cell formation was confirmed by both histological and proteome analyses, but the cellular response is less ordered and variable culminating in Soemmerring's ring (SR) formation and sometimes Elschnig's pearls. This we evidence for lenses from a single patient. No bow region or recognisable epithelial-fibre cell interface (EFI) was evident and consequently the fibre cells were disorganised. We conclude that lens cells require spatial and cellular cues to initiate, sustain and produce an optically functional tissue in addition to capsule integrity and the EFI.

Chlorella oil nanoliposomes (CO-NLP) were synthesized through ultrasonic injection with ethanol, and their physicochemical properties and hypolipidemic efficacy were systematically investigated. The results revealed that the mean particle size of CO-NLP was 86.90 nm and the encapsulation efficiency (EE) was 92.84%. Storage conditions at 4 °C were conducive to the stability of CO-NLP, maintaining an EE of approximately 90% even after 10 days of storage. The release profile of CO-NLP adhered more closely to the first-order kinetic model during in vitro assessments, exhibiting a slower release rate compared to free microalgae oil. In simulated in vitro digestion experiments, lipolytic reactions of CO-NLP were observed during intestinal digestion subsequent to nanoliposome administration. Notably, the inhibitory effect of CO-NLP on cholesterol esterase activity was measured at 85.42%. Additionally, the average fluorescence intensity of nematodes in the CO-NLP group was 52.17% lower than in the control group at a CO-NLP concentration of 500 μg/mL, which suggests a pronounced lipid-lowering effect of CO-NLP. Therefore, the CO-NLP exhibited characteristics of small and uniform particle size, elevated storage stability, gradual release during intestinal digestion, and a noteworthy hypolipidemic effect. These findings designate CO-NLP as a novel lipid-lowering active product, demonstrating potential for the development of functional foods.

Agonist-mediated signaling by the endothelium controls virtually all vascular functions. Because of the large diversity of agonists, each with varying concentrations, background noise often obscures individual cellular signals. How the endothelium distinguishes low-level fluctuations from noise and decodes and integrates physiologically relevant information remains unclear. Here, we recorded changes in intracellular Ca(2+) concentrations in response to acetylcholine in areas encompassing hundreds of endothelial cells from inside intact pressurized arteries. Individual cells responded to acetylcholine with a concentration-dependent increase in Ca(2+) signals spanning a single order of magnitude. Interestingly, however, intercellular response variation extended over 3 orders of magnitude of agonist concentration, thus crucially enhancing the collective bandwidth of endothelial responses to agonists. We also show the accuracy of this collective mode of detection is facilitated by spatially restricted clusters of comparably sensitive cells arising from heterogeneous receptor expression. Simultaneous stimulation of clusters triggered Ca(2+) signals that were transmitted to neighboring cells in a manner that scaled with agonist concentration. Thus, the endothelium detects agonists by acting as a distributed sensing system. Specialized clusters of detector cells, analogous to relay nodes in modern communication networks, integrate populationwide inputs, and enable robust noise filtering for efficient high-fidelity signaling.-Wilson, C., Saunter, C. D., Girkin, J. M., McCarron, J. G. Clusters of specialized detector cells provide sensitive and high fidelity receptor signaling in the intact endothelium.

The TRPV4 ion channels are Ca2+ permeable, non-selective cation channels that mediate large, but highly localized, Ca2+ signals in the endothelium. The mechanisms that permit highly localized Ca2+ changes to evoke cell-wide activity are incompletely understood. Here, we tested the hypothesis that TRPV4-mediated Ca2+ influx activates Ca2+ release from internal Ca2+ stores to generate widespread effects.

The feeling of hunger or satiety results from integration of the sensory nervous system with other physiological and metabolic cues. This regulates food intake, maintains homeostasis and prevents disease. In C. elegans, chemosensory neurons sense food and relay information to the rest of the animal via hormones to control food-related behaviour and physiology. Here we identify a new component of this system, SKN-1B which acts as a central food-responsive node, ultimately controlling satiety and metabolic homeostasis. SKN-1B, an ortholog of mammalian NF-E2 related transcription factors (Nrfs), has previously been implicated with metabolism, respiration and the increased lifespan incurred by dietary restriction. Here we show that SKN-1B acts in two hypothalamus-like ASI neurons to sense food, communicate nutritional status to the organism, and control satiety and exploratory behaviours. This is achieved by SKN-1B modulating endocrine signalling pathways (IIS and TGF-β), and by promoting a robust mitochondrial network. Our data suggest a food-sensing and satiety role for mammalian Nrf proteins.

Although excessive lipid accumulation is a hallmark of obesity-related pathologies, some lipids are beneficial. Oleic acid (OA), the most abundant monounsaturated fatty acid (FA), promotes health and longevity. Here, we show that OA benefits Caenorhabditis elegans by activating the endoplasmic reticulum (ER)-resident transcription factor SKN-1A (Nrf1/NFE2L1) in a lipid homeostasis response. SKN-1A/Nrf1 is cleared from the ER by the ER-associated degradation (ERAD) machinery and stabilized when proteasome activity is low and canonically maintains proteasome homeostasis. Unexpectedly, OA increases nuclear SKN-1A levels independently of proteasome activity, through lipid droplet-dependent enhancement of ERAD. In turn, SKN-1A reduces steatosis by reshaping the lipid metabolism transcriptome and mediates longevity from OA provided through endogenous accumulation, reduced H3K4 trimethylation, or dietary supplementation. Our findings reveal an unexpected mechanism of FA signal transduction, as well as a lipid homeostasis pathway that provides strategies for opposing steatosis and aging, and may mediate some benefits of the OA-rich Mediterranean diet.