Human Brain Microvascular Endothelial Cells Exposure to SARS-CoV-2 Leads to Inflammatory Activation through NF-κB Non-Canonical Pathway and Mitochondrial Remodeling.

Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the blood-brain barrier. Despite the low to non-productive viral replication, SARS-CoV-2-exposed cultures displayed increased immunoreactivity for cleaved caspase-3, an indicator of apoptotic cell death, tight junction protein expression, and immunolocalization. Transcriptomic profiling of SARS-CoV-2-challenged cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Low-dose lithium supplementation promotes adipose tissue browning and sarco(endo)plasmic reticulum Ca2+ ATPase uncoupling in muscle.

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) uncoupling in skeletal muscle and mitochondrial uncoupling via uncoupling protein 1 (UCP1) in brown/beige adipose tissue are two mechanisms implicated in energy expenditure. Here, we investigated the effects of glycogen synthase kinase 3 (GSK3) inhibition via lithium chloride (LiCl) treatment on SERCA uncoupling in skeletal muscle and UCP1 expression in adipose. C2C12 and 3T3-L1 cells treated with LiCl had increased SERCA uncoupling and UCP1 protein levels, respectively, ultimately raising cellular respiration; however, this was only observed when LiCl treatment occurred throughout differentiation. In vivo, LiCl treatment (10 mg/kg/day) increased food intake in chow-fed diet and high-fat diet (HFD; 60% kcal)-fed male mice without increasing body mass-a result attributed to elevated daily energy expenditure. In soleus muscle, we determined that LiCl treatment promoted SERCA uncoupling via increased expression of SERCA uncouplers, sarcolipin and/or neuronatin, under chow-fed and HFD-fed conditions. We attribute these effects to the GSK3 inhibition observed with LiCl treatment as partial muscle-specific GSK3 knockdown produced similar effects. In adipose, LiCl treatment inhibited GSK3 in inguinal white adipose tissue (iWAT) but not in brown adipose tissue under chow-fed conditions, which led to an increase in UCP1 in iWAT and a beiging-like effect with a multilocular phenotype. We did not observe this beiging-like effect and increase in UCP1 in mice fed a HFD, as LiCl could not overcome the ensuing overactivation of GSK3. Nonetheless, our study establishes novel regulatory links between GSK3 and SERCA uncoupling in muscle and GSK3 and UCP1 and beiging in iWAT.

SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non-productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non- productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Mitochondrial and metabolic remodelling in human skin fibroblasts in response to glucose availability.

Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine, and the validation of ingredients for cosmetics. Given these cells' short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in normal human dermal fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level [low glucose medium (LGm)], or its complete removal and replacement by galactose [medium that forces oxidative phosphorylation (OXPHOSm)], always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm was it possible to observe the selective inhibition of mitochondrial adenosine triphosphate (ATP) production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate and extracellular acidification rate, oxidation of citric acid cycle substrates, fatty acids, lactate, and other substrates, increased mitochondrial network extension and polarization, the increased protein content of voltage-dependent anion channel (VDAC) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha and changes in several key transcripts related to energy metabolism. LGm did not promote significant metabolic changes in NHDF, although mitochondrial network extension and VDAC protein content were increased compared to HGm-cultured cells. Our results indicate that short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.

Bioabsorbable metal zinc differentially affects mitochondria in vascular endothelial and smooth muscle cells.

Zinc is an essential trace element having various structural, catalytic and regulatory interactions with an estimated 3000 proteins. Zinc has drawn recent attention for its use, both as pure metal and alloyed, in arterial stents due to its biodegradability, biocompatibility, and low corrosion rates. Previous studies have demonstrated that zinc metal implants prevent the development of neointimal hyperplasia, which is a common cause of restenosis following coronary intervention. This suppression appears to be smooth muscle cell-specific, as reendothelization of the neointima is not inhibited. To better understand the basis of zinc's differential effects on rat aortic smooth muscle (RASMC) versus endothelial (RAENDO) cells, we conducted a transcriptomic analysis of both cell types following one-week continuous treatment with 5 µM or 50 µM zinc. This analysis indicated that genes whose protein products regulate mitochondrial functions, including oxidative phosphorylation and fusion/fission, are differentially affected by zinc in the two cell types. To better understand this, we performed Seahorse metabolic flux assays and quantitative imaging of mitochondrial networks in both cell types. Zinc treatment differently affected energy metabolism and mitochondrial structure/function in the two cell types. For example, both basal and maximal oxygen consumption rates were increased by zinc in RASMC but not in RAENDO. Zinc treatment increased apparent mitochondrial fusion in RASMC cells but increased mitochondrial fission in RAENDO cells. These results provide some insight into the mechanisms by which zinc treatment differently affects the two cell types and this information is important for understanding the role of zinc treatment in vascular cells and improving its use in biodegradable metal implants.

Hypoxia inducible factors as mediators of reactive oxygen/nitrogen species homeostasis in physiological normoxia.

Although once considered by biologists almost exclusively for their toxicity, reactive oxygen (ROS) and nitrogen (RNS) species produced within normal cells under baseline physiological conditions are now appreciated as redox regulators of a wide range of protein functions. Two families of enzymes, the NADPH oxidases (NOXs) and nitric oxide synthases (NOSs), are major sources of ROS/RNS from molecular oxygen. Aquaporins (AQPs) are membrane channels capable of transporting some ROS/RNS, in particular hydrogen peroxide and perhaps nitric oxide. The activities of all these enzymes and channels are sensitive to variations in oxygen levels within the physiological range experienced by cells in the human body. Since ROS/RNS have important physiological roles and their endogenous production is affected by oxygen levels, we hypothesize that the synthesis of these proteins is increased at lower oxygen levels within the physiological range of most human cells in vivo, i.e. 2-5%, in order to facilitate the maintenance of ROS/RNS production rates. We further postulate that this is achieved, at least in part, by transcriptional stimulation mediated by the activity of hypoxia inducible factors (HIFs), which are strongly regulated by oxygen levels over the same range of oxygen. Here we survey the evidence supporting this hypothesis, including induction of expression of NOXs, NOSs, and AQPs at lower oxygen levels, presence of hypoxia response elements in the corresponding human genes, and evidence from chromatin immunoprecipitation (ChIP) experiments that HIF-1 and/or HIF-2 bind these regions. We find a significant amount of empirical data supporting the hypothesis that HIFs could function as physiological regulators of ROS/RNS homeostasis in the normoxic range in human cells.

Hypoxio: a simple solution to preventing pericellular hypoxia in cell monolayers growing at physiological oxygen levels.

Culturing cells as adherent monolayers is a common approach in cell biology. For cell culture experiments to yield reliable results it is important to replicate in vivo conditions as faithfully as possible. Increasingly, researchers appreciate the importance of oxygen in cell physiology and the corresponding need to maintain physiologically relevant oxygen levels during experiments. However, although oxygen levels are sometimes monitored over the course of an experiment, this is virtually always in the incubator gas phase and not in the media bathing cells. When incubator oxygen levels are set to a physiologically appropriate level, typically 2-6%, the pericellular oxygen levels experienced by cells may be substantially lower, particularly under conditions where cells are respiring rapidly. We have developed a simple approach to prevent this problem. 'Hypoxio' is a software application that uses real time measurements of pericellular oxygen to coordinate media mixing via a tilt table. Hypoxio allows the user to set a threshold below which it initiates a mixing cycle of user-adjustable duration to abolish standing gradients associated with pericellular hypoxia. Here we describe Hypoxio, demonstrate its efficacy, and direct the reader to our GitHub site for downloadabale software and a description of hardware.