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Vascularization remains an obstacle when engineering complex tissues for regeneration and disease modeling. Although progress has been made in recreating 3D vascular structures, challenges exist in generating a mature, functional endothelium. It is demonstrated that perfusing engineered microvessels with platelet‐rich plasma, a critical homeostatic component in vivo that is often overlooked in vitro, substantially transforms the endothelium, both maturing endothelial cells and improving functionality in 24 h. Platelets readily adhered to the exposed collagen‐I substrate through small gaps within engineered vessels without forming thrombi. The adherent platelets improve barrier function, enhance endothelial glycolysis, reduce thrombogenicity, and enrich smooth muscle cell growth surrounding the endothelium. These findings demonstrate that platelets are essential to the function of endothelium during vascular maturation and remodeling. This study sheds light on a potential strategy to engineer stable, implantable vascular networks., Perfusion of platelet‐rich plasma (PRP) substantially transforms the endothelium in engineered microvessels in 24 h. PRP increases vessel diameter, improves barrier function, enhances endothelial glycolysis, reduces thrombogenicity, and enriches smooth muscle cell growth surrounding the endothelium. This work provides a potential strategy to improve vascular maturation and remodeling in engineered complex tissues for implantation and disease modeling.

Rationale: The mitochondrial permeability transition pore is a well-known initiator of cell death that is increasingly recognized as a physiological modulator of cellular metabolism. Objective: We sought to identify how the genetic deletion of a key regulatory subunit of the mitochondrial permeability transition pore, cyclophilin D (CypD), influenced endothelial metabolism and intracellular signaling. Methods and Results: In cultured primary human endothelial cells, genetic targeting of CypD using siRNA or shRNA resulted in a constitutive increase in mitochondrial matrix Ca 2+ and reduced nicotinamide adenine dinucleotide (NADH). Elevated matrix NADH, in turn, diminished the cytosolic NAD + /NADH ratio and triggered a subsequent downregulation of the NAD + -dependent deacetylase sirtuin 1 (SIRT1). Downstream of SIRT1, CypD-deficient endothelial cells exhibited reduced phosphatase and tensin homolog expression and a constitutive rise in the phosphorylation of angiogenic Akt. Similar changes in SIRT1, phosphatase and tensin homolog, and Akt were also noted in the aorta and lungs of CypD knockout mice. Functionally, CypD-deficient endothelial cells and aortic tissue from CypD knockout mice exhibited a dramatic increase in angiogenesis at baseline and when exposed to vascular endothelial growth factor. The NAD + precursor nicotinamide mononucleotide restored the cellular NAD + /NADH ratio and normalized the CypD-deficient phenotype. CypD knockout mice also presented accelerated wound healing and increased neovascularization on tissue injury as monitored by optical microangiography. Conclusions: Our study reveals the importance of the mitochondrial permeability transition pore in the regulation of endothelial mitochondrial metabolism and vascular function. The mitochondrial regulation of SIRT1 has broad implications in the epigenetic regulation of endothelial phenotype.

Summary The endothelium first forms in the blood islands in the extra-embryonic yolk sac and then throughout the embryo to establish circulatory networks that further acquire organ-specific properties during development to support diverse organ functions. Here, we investigated the properties of endothelial cells (ECs), isolated from four human major organs—the heart, lung, liver, and kidneys—in individual fetal tissues at three months' gestation, at gene expression, and at cellular function levels. We showed that organ-specific ECs have distinct expression patterns of gene clusters, which support their specific organ development and functions. These ECs displayed distinct barrier properties, angiogenic potential, and metabolic rate and support specific organ functions. Our findings showed the link between human EC heterogeneity and organ development and can be exploited therapeutically to contribute in organ regeneration, disease modeling, as well as guiding differentiation of tissue-specific ECs from human pluripotent stem cells., Graphical Abstract, Highlights • Isolate, culture, and characterize four human organ-specific endothelial cells • Identify and validate heritable gene expression clusters in human organ-specific ECs • Uncover the contribution of organ-specific ECs on vascular and organ function, Stem Cells Research; Developmental Biology; Biology of Human Development

Angiogenesis is a dynamic and energy-consuming process, requiring endothelial cells to switch from a quiescent state to a migratory and proliferative phenotype in order to support the formation of new blood vessels. Despite their proximity to oxygenated blood endothelial cells are adept at utilizing glycolysis as an energy source to the detriment of mitochondrial oxidative phosphorylation. In this context, endothelial mitochondria have emerged as signaling hubs that modulate a wide range of endothelial functions, including angiogenesis, by coordinating reactive oxygen species and calcium signaling, metabolism and apoptosis. In this chapter we present an overview of the mitochondrial functions implicated in promoting or hindering the angiogenic capacity of endothelial cells, with emphasis on the mitochondrial proteins directly linked to angiogenesis. We also focus on recent findings identifying mitochondrial targeting compounds that exhibit pro-angiogenic or anti-angiogenic properties, and could therefore be of clinical importance for the treatment of vascular pathologies.

Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD(+)/NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades.

The isothiourea derivative, KB-R7943, inhibits the reverse-mode of the plasma membrane sodium/calcium exchanger and protects against ischemia/reperfusion injury. The mechanism through which KB-R7943 confers protection, however, remains controversial. Recently, KB-R7943 has been shown to inhibit mitochondrial calcium uptake and matrix overload, which may contribute to its protective effects. While using KB-R7943 for this purpose, we find here no evidence that KB-R7943 directly blocks mitochondrial calcium uptake. Rather, we find that KB-R7943 inhibits opening of the mitochondrial permeability transition pore in permeabilized cells and isolated liver mitochondria. Furthermore, we find that this observation correlates with protection against calcium ionophore-induced mitochondrial membrane potential depolarization and cell death, without detrimental effects to basal mitochondrial membrane potential or complex I-dependent mitochondrial respiration. Our data reveal another mechanism through which KB-R7943 may protect against calcium-induced injury, as well as a novel means to inhibit the mitochondrial permeability transition pore.

Angiogenesis is a dynamic and energy-consuming process, requiring endothelial cells to switch from a quiescent state to a migratory and proliferative phenotype in order to support the formation of new blood vessels. Despite their proximity to oxygenated blood endothelial cells are adept at utilizing glycolysis as an energy source to the detriment of mitochondrial oxidative phosphorylation. In this context, endothelial mitochondria have emerged as signaling hubs that modulate a wide range of endothelial functions, including angiogenesis, by coordinating reactive oxygen species and calcium signaling, metabolism and apoptosis. In this chapter we present an overview of the mitochondrial functions implicated in promoting or hindering the angiogenic capacity of endothelial cells, with emphasis on the mitochondrial proteins directly linked to angiogenesis. We also focus on recent findings identifying mitochondrial targeting compounds that exhibit pro-angiogenic or anti-angiogenic properties, and could therefore be of clinical importance for the treatment of vascular pathologies.

Cancer cells show an abnormal balance of reduced/oxidised species and the detection of cancer redox balance can be exploited for diagnostic purposes. In particular, it is known that the expression of oncogenic Ras changes the intracellular oxidised/reduced glutathione balance. Electrochemical imaging of living cells is currently feasible by scanning electrochemical microscopy and was used here, using ferrocenemethanol as redox mediator, to measure the oxidised/reduced glutathione balance in human breast epithelial MCF10A cells expressing constitutively active Ha-Ras Val12 mutant compared to normal MCF10A cells. Oxidized ferrocenemethanol is reduced by glutathione and the resulting maps of current over cell cultures were different for transformed cells compared to normal cells. Furthermore, scanning electrochemical microscopy using ferrocenemethanol redox mediator was able to distinguish human lung carcinoma cells from the surrounding normal epithelium of clinical specimens. We propose that scanning electrochemical microscopy analysis using specific redox mediator such as ferrocenemethanol may become a useful tool in cancer diagnostics.

Mitochondrial calcium uptake stimulates bioenergetics and drives energy production in metabolic tissue. It is unknown how a calcium-mediated acceleration in matrix bioenergetics would influence cellular metabolism in glycolytic cells that do not require mitochondria for ATP production. Using primary human endothelial cells (ECs), we discovered that repetitive cytosolic calcium signals (oscillations) chronically loaded into the mitochondrial matrix. Mitochondrial calcium loading in turn stimulated bioenergetics and a persistent elevation in NADH. Rather than serving as an impetus for mitochondrial ATP generation, matrix NADH rapidly transmitted to the cytosol to influence the activity and expression of cytosolic sirtuins, resulting in global changes in protein acetylation. In endothelial cells, the mitochondrion-driven reduction in both the cytosolic and mitochondrial NAD(+)/NADH ratio stimulated a compensatory increase in SIRT1 protein levels that had an anti-inflammatory effect. Our studies reveal the physiologic importance of mitochondrial bioenergetics in the metabolic regulation of sirtuins and cytosolic signaling cascades.

Mitochondrial respiration produces both complete and partially reduced oxygen species that are involved in physiological and pathological processes. Indeed, unspecific oxidative damage induced by excessive mitochondrial reactive oxygen species (ROS) plays a role in aging and several diseases, whereas low amounts of ROS act in physiological signaling processes. The exact molecular species, the rate, and the conditions of mitochondrial ROS release are not clearly evaluable by current methods based on oxidation sensitive markers. Recently, electrochemical analysis of biological samples has improved. Following latest methodology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism in isolated mitochondria through simultaneous measurement of O2 consumption and H2O2 production. Our experiments confirm active H2O2 production by respiring mouse liver mitochondria and show that ATP synthase activation increases the rate of H2O2, suggesting that state 3 mitochondria might induce the cell through oxidative signals.

Mitochondrial respiration produces both complete and partially reduced oxygen species that are involved in physiological and pathological processes. Indeed, unspecific oxidative damage induced by excessive mitochondrial reactive oxygen species (ROS) plays a role in aging and several diseases, whereas low amounts of ROS act in physiological signaling processes. The exact molecular species, the rate, and the conditions of mitochondrial ROS release are not clearly evaluable by current methods based on oxidation sensitive markers. Recently, electrochemical analysis of biological samples has improved. Following latest methodology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism in isolated mitochondria through simultaneous measurement of O2 consumption and H2O2 production. Our experiments confirm active H2O2 production by respiring mouse liver mitochondria and show that ATP synthase activation increases the rate of H2O2, suggesting that state 3 mitochondria might induce the cell through oxidative signals.

Reactive oxygen species (ROS) are regarded as hazardous by-products of mitochondrial respiration. In addition to the respiratory chain, specific ROS-generating systems have evolved. In particular, p66Shc is a mitochondrial redox protein that oxidizes cytochrome c to generate H2O2. Consistently, the deletion of p66Shc in cells and tissue results in reduced levels of ROS and oxidative stress. Taking advantage of the p66Shc knock out (p66KO) mouse model of decreased ROS production, we assessed the role of endogenously-produced ROS in tumorigenesis. Spontaneous tumor incidence was investigated and found unaltered in two different strains, 129Sv and C57Bl/6J, p66KO mice. In addition, papilloma formation upon exposure to ultraviolet radiation (UV) or 7,12-Dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol- 13-acetate (DMBA/TPA) was found to be slightly lower in the absence of p66Shc. The role of p66Shc in tumorigenesis was also investigated in the absence of the tumor suppressor gene p53 (p53KO) by generating p53-p66Shc double knock out (DKO) mice. Notably, DKO mice displayed a significantly increased lifespan compared to p53KO mice. In addition, 2-deoxy-2-(18F)fluoro-D-glucose Positron Emission Tomography ([18F]FDG PET) analysis allowed to determine that disease onset occurred later in life in DKO mice compared to p53KO and that a low percentage of these mice did not develop tumors. Overall, these results indicate that although tumor incidence is not decreased in p66KO mice, p66Shc contributes to tumor initiation, in particular upon activation by carcinogens as well as when p53- mediated tumor suppression mechanisms defect.

Mitochondrial respiration generates reactive oxygen species that are involved in physiological and pathological processes. The majority of methods, with exception of electron paramagnetic resonance, used to evaluate the identity, the rate and the conditions of the reactive oxygen species produced by mitochondria, are mainly based on oxidation sensitive markers. Following latest electrochemical methodology, we implemented a novel electrochemical assay for the investigation of aerobic metabolism in preparations of isolated mitochondria through simultaneous measurement of O2 consumption and reactive species production. This electrochemical assay reveals active H2O2 production by respiring mouse liver mitochondria, and shows that ATP synthase activation and moderate depolarization increase the rate of H2O2 formation, suggesting that ATP synthesizing (state 3) mitochondria might contribute to oxidative stress or signaling.

A novel method is proposed for using scanning electrochemical microscopy to draw pre-defined patterns of reactive spots onto conducting substrates that may then be functionalized—ad hoc and at will—with any molecular species containing an amine or other nucleophilic group, including proteins and enzymes.