Oxidative injury to cells such as the retinal pigment epithelium (RPE) is often modeled using H 2 O 2 -treated cultures, but H 2 O 2 concentrations are not sustained in culture medium. Here medium levels of H 2 O 2 and cytotoxicity were analyzed in ARPE-19 cultures following H 2 O 2 delivery as a single pulse or with continuous generation using glucose oxidase (GOx). When added as a pulse, H 2 O 2 is rapidly depleted (within 2 hr); cytotoxicity at 24, determined by the MTT assay for mitochondrial function, is unaffected by medium replacement at 2 hr. Continuous generation of H 2 O 2 produces complex outcomes. At low GOx concentrations, H 2 O 2 levels are sustained by conditions in which generation matches depletion, but when GOx concentrations produce cytotoxic levels of H 2 O 2 , oxidant depletion accelerates. Acceleration results partly from the release of contents from oxidant damaged cells as indicated by testing depletion after controlled membrane disruption with detergents. Cytotoxicity analyses show that cells can tolerate short exposure to high H 2 O 2 doses delivered as a pulse but are susceptible to lower chronic doses. The results provide broadly applicable guidance for using GOx to produce sustained H 2 O 2 levels in cultured cells. This approach will be specifically useful for modeling chronic stress relevant for RPE aging and have wider value for studying cellular effects of sub-lethal oxidant injury and for evaluating antioxidants that may protect significantly against mild but not lethal stress.

Carbon monoxide (CO), a product of heme degradation by heme oxygenases, plays an important role in vascular homeostasis. Recent evidence indicates that mitochondria are among a number of molecular targets that mediate the cellular actions of CO. In the present study we characterized the effects of CO released from CORM-401 on mitochondrial respiration and glycolysis in intact human endothelial cells using electron paramagnetic resonance (EPR) oximetry and the Seahorse XF technology. We found that CORM-401 (10-100μM) induced a persistent increase in the oxygen consumption rate (OCR) that was accompanied by inhibition of glycolysis (extracellular acidification rate, ECAR) and a decrease in ATP-turnover. Furthermore, CORM-401 increased proton leak, diminished mitochondrial reserve capacity and enhanced non-mitochondrial respiration. Inactive CORM-401 (iCORM-401) neither induced mitochondrial uncoupling nor inhibited glycolysis, supporting a direct role of CO in the endothelial metabolic response induced by CORM-401. Interestingly, blockade of mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBKCa) with paxilline abolished the increase in OCR promoted by CORM-401 without affecting ECAR; patch-clamp experiments confirmed that CO derived from CORM-401 activated mitoBKCa channels present in mitochondria. Conversely, stabilization of glycolysis by MG132 prevented CORM-401-mediated decrease in ECAR but did not modify the OCR response. In summary, we demonstrated in intact endothelial cells that CO induces a two-component metabolic response: uncoupling of mitochondrial respiration dependent on the activation of mitoBKCa channels and inhibition of glycolysis independent of mitoBKCa channels.

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Healthy liver sinusoidal endothelial cells (LSECs) maintain liver homeostasis, while LSEC dysfunction was suggested to coincide with defenestration. Here, we have revisited the relationship between LSEC pro-inflammatory response, defenestration, and impairment of LSEC bioenergetics in non-alcoholic fatty liver disease (NAFLD) in mice. We characterized inflammatory response, morphology as well as bioenergetics of LSECs in early and late phases of high fat diet (HFD)-induced NAFLD. LSEC phenotype was evaluated at early (2–8 week) and late (15–20 week) stages of NAFLD progression induced by HFD in male C57Bl/6 mice. NAFLD progression was monitored by insulin resistance, liver steatosis and obesity. LSEC phenotype was determined in isolated, primary LSECs by immunocytochemistry, mRNA gene expression (qRT-PCR), secreted prostanoids (LC/MS/MS) and bioenergetics (Seahorse FX Analyzer). LSEC morphology was examined using SEM and AFM techniques. Early phase of NAFLD, characterized by significant liver steatosis and prominent insulin resistance, was related with LSEC pro-inflammatory phenotype as evidenced by elevated ICAM-1, E-selectin and PECAM-1 expression. Transiently impaired mitochondrial phosphorylation in LSECs was compensated by increased glycolysis. Late stage of NAFLD was featured by prominent activation of pro-inflammatory LSEC phenotype (ICAM-1, E-selectin, PECAM-1 expression, increased COX-2, IL-6, and NOX-2 mRNA expression), activation of pro-inflammatory prostaglandins release (PGE 2 and PGF 2α ) and preserved LSEC bioenergetics. Neither in the early nor in the late phase of NAFLD, were LSEC fenestrae compromised. In the early and late phases of NAFLD, despite metabolic and pro-inflammatory burden linked to HFD, LSEC fenestrae and bioenergetics are functionally preserved. These results suggest prominent adaptive capacity of LSECs that might mitigate NAFLD progression.

Objectives: Carbon monoxide (CO) produced by haem oxygenases or released by CO releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO–induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry–based metabolomics were used. CORM-A1–induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1–induced delay of Krebs cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD + ) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD + –dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1–induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD + regeneration, pointing out to the key role of NAD + depletion in the inhibition of platelet bioenergetics by CORM-A1. Conclusions: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration—attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis—ascribed to cytosolic NAD+ depletion.

Summary Melanosomes were recently shown to protect ARPE‐19 cells, a human retinal pigment epithelium (RPE) cell line, against oxidative stress induced by hydrogen peroxide. One postulated mechanism of antioxidant action of melanin is its ability to bind metal ions. The aim here was to determine whether melanosomes are competent to bind iron within living cells, exhibiting a property previously shown only in model systems. The outcomes indicate retention of prebound iron and accumulation of iron by granules after iron delivery to cells via the culture medium, as determined by both colorimetric and electron spin resonance analyses for bound‐to‐melanosome iron. Manipulation of iron content did not affect the pigment's ability to protect cells against H2O2, but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.

Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that β1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer–endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.

Nrf2 is a master regulator of antioxidant cellular defence, and agents activating the Nrf2 pathway have been tested in various diseases. However, unexpected side effects of cardiovascular nature reported for bardoxolone methyl in patients with type 2 diabetes mellitus and stage 4 chronic kidney disease (the BEACON trial) still have not been fully explained. Here, we aimed to characterize the effects of bardoxolone methyl compared with other Nrf2 activators—dimethyl fumarate and L-sulforaphane—on human microvascular endothelium. Endothelial toxicity, bioenergetics, mitochondrial membrane potential, endothelin-1 (ET-1) release, endothelial permeability, Nrf2 expression, and ROS production were assessed in human microvascular endothelial cells (HMEC-1) incubated for 3 and 24 hours with 100 nM–5 μM of either bardoxolone methyl, dimethyl fumarate, or L-sulforaphane. Three-hour incubation with bardoxolone methyl (100 nM–5 μM), although not toxic to endothelial cells, significantly affected endothelial bioenergetics by decreasing mitochondrial membrane potential ( concentrations ≥ 3   μ M ), decreasing spare respiratory capacity ( concentrations ≥ 1   μ M ), and increasing proton leak ( concentrations ≥ 500   nM ), while dimethyl fumarate and L-sulforaphane did not exert such actions. Bardoxolone methyl at concentrations ≥ 3   μ M also decreased cellular viability and induced necrosis and apoptosis in the endothelium upon 24-hour incubation. In turn, endothelin-1 decreased permeability in endothelial cells in picomolar range, while bardoxolone methyl decreased ET-1 release and increased endothelial permeability even after short-term (3 hours) incubation. In conclusion, despite that all three Nrf2 activators exerted some beneficial effects on the endothelium, as evidenced by a decrease in ROS production, bardoxolone methyl, the most potent Nrf2 activator among the tested compounds, displayed a distinct endothelial profile of activity comprising detrimental effects on mitochondria and cellular viability and suppression of endothelial ET-1 release possibly interfering with ET-1–dependent local regulation of endothelial permeability.