Endosomes: Guardians Against [Ru(Phen)3]2+ Photo-action In Endothelial Cells During In Vivo pO2 Detection?

Phototoxicity is a side-effect of in vitro and in vivo oxygen partial pressure (pO(2)) detection by luminescence lifetime measurement methods. Dichlorotris(1,10-phenanthroline)-ruthenium(II) hydrate (Ru(Phen)(3)) is a water soluble pO(2) probe associated with low phototoxicity, which we investigated in vivo in the chick's chorioallantoic membrane (CAM) after intravenous or topical administration and in vitro in normal human coronary artery endothelial cells (HCAEC). In vivo, the level of intravenously injected Ru(Phen)(3) decreases within several minutes, whereas the maximum of its biodistribution is observed during the first 2 h after topical application. Both routes are followed by convergence to almost identical "intra/extra-vascular" levels of Ru(Phen)(3). In vitro, we observed that Ru(Phen)(3) enters cells via endocytosis and is then redistributed. None of the studied conditions induced modification of lysosomal or mitochondrial membranes without illumination. No nuclear accumulation was observed. Without illumination Ru(Phen)(3) induces changes in endoplasmic reticulum (ER)-to-Golgi transport. The phototoxic effect of Ru(Phen)(3) leads to more marked ultrastructural changes than administration of Ru(Phen)(3) only (in the dark). These could lead to disruption of Ca2+ homeostasis accompanied by mitochondrial changes or to changes in secretory pathways. In conclusion, we have demonstrated that the intravenous injection of Ru(Phen)(3) into the CAM model mostly leads to extracellular localization of Ru(Phen)(3), while its topical application induces intracellular localization. We have shown in vivo that Ru(Phen)(3) induces minimal photo-damage after illumination with light doses larger by two orders of magnitude than those used for pO(2) measurements. This low phototoxicity is due to the fact that Ru(Phen)(3) enters endothelial cells via endocytosis and is then redistributed towards peroxisomes and other endosomal and secretory vesicles before it is eliminated via exocytosis. Cellular response to Ru(Phen)(3), survival or death, depends on its intracellular concentration and oxidation-reduction properties.

In vivo measurement of tissue oxygenation by time-resolved luminescence spectroscopy: advantageous properties of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate

Sandrine Gay, Veronika Huntosova, Patrycja Nowak-Sliwinska, Senthil Kumar Rajendran, Hubert van den Bergh, Georges Wagnières, Matthieu Zellweger

Measuring tissue oxygenation in vivo is of interest in fundamental biological as well as medical applications. One minimally invasive approach to assess the oxygen partial pressure in tissue (pO(2)) is to measure the oxygen-dependent luminescence lifetime of molecular probes. The relation between tissue pO(2) and the probes' luminescence lifetime is governed by the Stern-Volmer equation. Unfortunately, virtually all oxygen-sensitive probes based on this principle induce some degree of phototoxicity. For that reason, we studied the oxygen sensitivity and phototoxicity of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate [Ru(Phen)] using a dedicated optical fiber-based, time-resolved spectrometer in the chicken embryo chorioallantoic membrane. We demonstrated that, after intravenous injection, Ru(Phen)'s luminescence lifetime presents an easily detectable pO(2) dependence at a low drug dose (1 mg/kg) and low fluence (120 mJ/cm(2) at 470 nm). The phototoxic threshold was found to be at 10 J/cm(2) with the same wavelength and drug dose, i.e., about two orders of magnitude larger than the fluence necessary to perform a pO(2) measurement. Finally, an illustrative application of this pO(2) measurement approach in a hypoxic tumor environment is presented. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)

Spie-Soc Photo-Optical Instrumentation Engineers2014

Dissecting the role of the mitochondrial carrier SLC25A47

Nadia Bresciani

Transporters of the SLC25 mitochondrial carrier superfamily bridge cytoplasmic and mitochondrial metabolism by channeling metabolites across mitochondrial membranes and are pivotal for metabolic homeostasis. Despite their physiological relevance as gatekeepers of cellular metabolism, most of the SLC25 family members remain uncharacterized. During my PhD studies, I investigated the function of SLC25A47, an orphan carrier uniquely expressed in hepatocytes. We used a murine loss-of-function (LOF) model to unravel the role of this transporter in mitochondrial and hepatic homeostasis. Slc25a47hep-/- mice displayed a wide variety of metabolic abnormalities, as a result of sustained energy deficiency in the liver originating from impaired mitochondrial respiration in this organ. This mitochondrial phenotype was associated with a robust activation of the mitochondrial stress response (MSR) in the liver, which in turn, induced the secretion of several mitokines, amongst which FGF21 played a preponderant role in the translation of the effects of the MSR on systemic physiology. To dissect the FGF21-dependent and -independent physiological changes induced by the loss of Slc25a47, we generated a double Slc25a47-Fgf21 LOF mouse model, and demonstrated that several aspects of the hypermetabolic state were entirely driven by hepatic secretion of FGF21. On the other hand, the metabolic fuel inflexibility induced by loss of Slc25a47 could not be rescued by genetic removal of Fgf21. Finally, we challenged Slc25a47hep-/- mice with high-fat high-sucrose (HFHS) diet and observed the development of liver fibrosis.Collectively, the data presented in this thesis show that SLC25A47 is a novel determinant of hepatic metabolism and place this carrier at the center of mitochondrial homeostasis.

EPFL2022