Expanding the Reactive Sulfur Metabolome: Intracellular and Efflux Measurements of Small Oxoacids of Sulfur (SOS) and H2S in Human Primary Vascular Cell Culture.

Hydrogen sulfide (H2S) is an endogenous signaling molecule which is important for cardiovascular health, but its mechanism of action remains poorly understood. Here, we report measurements of H2S as well as its oxidized metabolites, termed small oxoacids of sulfur (SOS = HSOH and HOSOH), in four human primary vascular cell lines: smooth muscle and endothelial cells derived from both human arterial and coronary tissues. We use a methodology that targets small molecular weight sulfur species; mass spectrometric analysis allows for species quantification to report cellular concentrations based on an H2S calibration curve. The production of H2S and SOS is orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar). In all the primary lines measured, the distributions of these three species were HOSOH >H2S > HSOH, with much higher SOS than seen previously in non-vascular cell lines. H2S and SOS were effluxed from smooth muscle cells in higher concentrations than endothelial cells. Aortic smooth muscle cells were used to examine changes under hypoxic growth conditions. Hypoxia caused notable increases in HSOH and ROS, which we attribute to enhanced sulfide quinone oxidase activity that results in reverse electron transport.

Hyperoxia impairs induced pluripotent stem cell-derived endothelial cells and drives an atherosclerosis-like transcriptional phenotype.

Background: Induced pluripotent stem cells (iPSCs) directed to endothelial identity (iPSC-ECs) are emerging as a potent tool for regenerative medicine in vascular disease. However, iPSC-ECs lose expression of key identity markers under standard in vitro conditions, limiting their clinical applications. Methods: To model physiological in vivo conditions, we examined the bioenergetics, presence of key cell markers, and proliferative and angiogenic capacity in iPSC-ECs at late and early passage under hyperoxic (21%) and physiological (4%) oxygen concentrations. Results: Physoxia resulted in relative preservation of mitochondrial bioenergetic activity, as well as CD144 expression in late passage iPSC-ECs, but not proliferative capacity or tube formation. Single cell RNA sequencing (scRNA-seq) revealed that late passage hyperoxic iPSC-ECs develop an endothelial-to-mesenchymal phenotype. Comparing scRNA-seq data from iPSC-ECs and from atherosclerotic ECs revealed overlap of their transcriptional phenotypes. Conclusions: Taken together, our studies demonstrate that physiological 4% oxygen culture conditions were sufficient to improve mitochondrial function in high passage cells, but alone was insufficient to preserve angiogenic capacity. Furthermore, late passage cells under typical conditions take on an endothelial-to-mesenchymal phenotype with similarities to ECs found in atherosclerosis.

Myoglobinemia, Peripheral Arterial Disease, and Patient Mortality.

BACKGROUND: Peripheral arterial disease (PAD) causes leg muscle damage due to inadequate perfusion and increases cardiovascular events and mortality 2- to 3-fold. It is unclear if PAD is a biomarker for high-risk cardiovascular disease or if skeletal muscle injury harms arterial health. The objective of this work is to test if serum myoglobin levels (myoglobinemia) are a marker of PAD, and if so, whether myoglobin impairs vascular health. STUDY DESIGN: Patient blood samples were collected from PAD and control (no PAD) patients and interrogated for myoglobin concentrations and nitric oxide bioavailability. Patient mortality over time was captured from the medical record. Myoglobin activity was tested on endothelial cells and arterial function. RESULTS: Myoglobin is a biomarker for symptomatic PAD and was inversely related to nitric oxide bioavailability; 200 ng/mL myoglobin in vitro increased endothelial cell permeability in vitro and decreased nitrate bioavailability. Ex vivo, 100 ng/mL myoglobin increased vascular tone in naive murine aortas approximately 1.5 times, impairing absolute vessel relaxation. In vivo, we demonstrated that myoglobinemia caused impaired flow-mediated dilation in a porcine model. Patients presenting with myoglobin levels of 100 ng/mL or greater had significantly more deaths than those with myoglobin levels of less than 100 ng/mL. CONCLUSIONS: Using a combination of patient data, in vitro, ex vivo, and in vivo testing, we found that myoglobin is a biomarker for symptomatic PAD and a potent regulator of arterial health that can increase vascular tone, increase vascular permeability, and cause endothelial dysfunction, all of which may contribute to the vulnerability of PAD patients to cardiovascular events and death.

The ionophore thiomaltol induces rapid lysosomal accumulation of copper and apoptosis in melanoma.

In this report, we investigate the toxicity of the ionophore thiomaltol (Htma) and Cu salts to melanoma. Divalent metal complexes of thiomaltol display toxicity against A375 melanoma cell culture resulting in a distinct apoptotic response at submicromolar concentrations, with toxicity of Cu(tma)2 > Zn(tma)2 >> Ni(tma)2. In metal-chelated media, Htma treatment shows little toxicity, but the combination with supplemental CuCl2, termed Cu/Htma treatment, results in toxicity that increases with suprastoichiometric concentrations of CuCl2 and correlates with the accumulation of intracellular copper. Electron microscopy and confocal laser scanning microscopy of Cu/Htma treated cells shows a rapid accumulation of copper within lysosomes over the course of hours, concurrent with the onset of apoptosis. A buildup of ubiquitinated proteins due to proteasome inhibition is seen on the same timescale and correlates with increases of copper without additional Htma.

Characterization of Endogenous and Extruded H2S and Small Oxoacids of Sulfur (SOS) in Cell Cultures.

This report characterizes and quantifies endogenous hydrogen sulfide (H2S) and small oxoacids of sulfur (SOS = HOSH, HOSOH) in a panel of cell lines including human cancer (A375 melanoma cells, HeLa cervical cells) and noncancer (HEK293 embryonic kidney cells), as well as E. coli DH5α and S. cerevisiae S288C. The methodology used is a translation of well-studied nucleophilic and electrophilic traps for cysteine and oxidized cysteines residues to target small molecular weight sulfur species; mass spectrometric analysis allows for species quantification. The observed intracellular concentrations of H2S and SOS vary in different cell types, from nanomolar to femtomolar, typically with H2S > HOSOH > HOSH. We propose the term sulfome, a subset of the metabolome, describing the nonproteinaceous metabolites of H2S; the sulfomic index is as a measure of the S-oxide redox status, which gives a profile of endogenous sulfur at different oxidation states. An important observation is that H2S and SOS were found to be continuously extruded into surrounding media against a concentration gradient, implying an active efflux process. Small molecule inhibition of several H2S generating enzymes suggest that SOS are not derived solely from H2S oxidation. Even after successful inhibition of H2S production, cells maintain constant efflux and repopulate H2S and SOS over time. This work proves that these small sulfur oxoacids are generated in cells of all types, and their efflux implies that they play a role in cell signaling and possibly other vascular physiology attributed to H2S.