Heterozygous RPA2 variant as a novel genetic cause of telomere biology disorders.

Premature telomere shortening or telomere instability is associated with a group of rare and heterogeneous diseases collectively known as telomere biology disorders (TBDs). Here we identified two unrelated individuals with clinical manifestations of TBDs and short telomeres associated with the identical monoallelic variant c.767A>G; Y256C in RPA2 Although the replication protein A2 (RPA2) mutant did not affect ssDNA binding and G-quadruplex-unfolding properties of RPA, the mutation reduced the affinity of RPA2 with the ubiquitin ligase RFWD3 and reduced RPA ubiquitination. Using engineered knock-in cell lines, we found an accumulation of RPA at telomeres that did not trigger ATR activation but caused short and dysfunctional telomeres. Finally, both patients acquired, in a subset of blood cells, somatic genetic rescue events in either POT1 genes or TERT promoters known to counteract the accelerated telomere shortening. Collectively, our study indicates that variants in RPA2 represent a novel genetic cause of TBDs. Our results further support the fundamental role of the RPA complex in regulating telomere length and stability in humans.

mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

Vascular endothelial function masks increased sympathetic vasopressor activity in rats with metabolic syndrome.

Sympathetic hyperactivation, a common feature of obesity and metabolic syndrome, is a key trigger of hypertension. However, some obese subjects with autonomic imbalance present a dissociation between sympathetic activity-mediated vasoconstriction and increased blood pressure. Here, we aimed to determine in a rat model of metabolic syndrome whether the endothelium endothelial nitric oxide (NO) synthase (eNOS)-NO pathway contributes to counteract the vasopressor effect of the sympathetic system. Rats were fed a high-fat and high-sucrose (HFS) diet for 15 wk. Sympathovagal balance was evaluated by spectral analysis of heart rate variability and plasmatic catecholamine measurements. Blood pressure was measured in the presence or absence of N-nitro-l-arginine methyl ester (l-NAME) to inhibit the contribution of eNOS. Vascular reactivity was assessed on isolated aortic rings in response to α1-adrenergic agonist. The HFS diet increased sympathetic tone, which is characterized by a higher low on the high-frequency spectral power ratio and a higher plasmatic concentration of epinephrine. Despite this, no change in blood pressure was observed. Interestingly, HFS rats exhibited vascular hyporeactivity (-23.6%) to α1-adrenergic receptor stimulation that was abolished by endothelial removal or eNOS inhibition (l-NAME). In addition, eNOS phosphorylation (Ser1177) was increased in response to phenylephrine in HFS rats only. Accordingly, eNOS inhibition in vivo revealed higher blood pressure in HFS rats compared with control rats (147 vs. 126 mmHg for mean blood pressure, respectively). Restrain of adrenergic vasopressor action by endothelium eNOS is increased in HFS rats and contributes to maintained blood pressure in the physiological range. NEW & NOTEWORTHY Despite the fact that prohypertensive sympathetic nervous system activity is markedly increased in rats with early metabolic syndrome, they present with normal blood pressure. These observations appear to be explained by increased endothelial nitric oxide synthase response to adrenergic stimulation, which results in vascular hyporeactivity to α-adrenergic stimulation, and therefore blood pressure is preserved in the physiological range. Listen to this article's corresponding podcast at http://www.physiology.org/doi/10.1152/ajpheart.00217.2017 .

Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model.

Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas' leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5-12 µM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.

Increased Sirt1 secreted from visceral white adipose tissue is associated with improved glucose tolerance in obese Nrf2-deficient mice.

Obesity is associated with metabolic dysregulation characterized by insulin resistance and glucose intolerance. Nuclear factor E2-related factor (Nrf2) is a critical regulator of the stress response and Nrf2-deficient mice (Nrf2-/-) are protected against high fat diet (HFD)-induced metabolic derangement. We searched for factors that could underline this favorable phenotype and found that Nrf2-/- mice exhibit higher circulating levels of sirtuin 1 (Sirt1), a key player in cellular homeostasis and energy metabolism, compared to wild-type mice. Increased Sirt1 levels in Nrf2-/- mice were found not only in animals under standard diet but also following HFD. Interestingly, we report here that the visceral adipose tissue (eWAT) is the sole source of increased Sirt1 protein in plasma. eWAT and other fat depots displayed enhanced adipocytes lipolysis, increased fatty acid oxidation and glycolysis, suggesting autocrine and endocrine actions of Sirt1 in this model. We further demonstrate that removal of eWAT completely abolishes the increase in circulating Sirt1 and that this procedure suppresses the beneficial effect of Nrf2 deficiency on glucose tolerance, but not insulin sensitivity, following a HFD regime. Thus, in contrast to many other stressful conditions where Nrf2 deficiency exacerbates damage, our study indicates that up-regulation of Sirt1 levels specifically in the visceral adipose tissue of Nrf2-/- mice is a key adaptive mechanism that mitigates glucose intolerance induced by nutritional stress.

Adipose tissue senescence is mediated by increased ATP content after a short-term high-fat diet exposure.

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high-fat diet (HFD, 1-10 weeks) in 5-month-old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence-associated ß-galactosidase activity and cyclin-dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD-derived preadipocytes, as compared with chow diet-derived preadipocytes. One-month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD-induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.

LIPE-related lipodystrophic syndrome: clinical features and disease modeling using adipose stem cells.

OBJECTIVE: The term Multiple Symmetric Lipomatosis (MSL) describes a heterogeneous group of rare monogenic disorders and multifactorial conditions, characterized by upper-body adipose masses. Biallelic variants in LIPE encoding hormone-sensitive lipase (HSL), a key lipolytic enzyme, were implicated in three families worldwide. We aimed to further delineate LIPE-related clinical features and pathophysiological determinants. METHODS: A gene panel was used to identify pathogenic variants. The disease features were reviewed at the French lipodystrophy reference center. The immunohistological, ultrastructural, and protein expression characteristics of lipomatous tissue were determined in surgical samples from one patient. The functional impact of variants was investigated by developing a model of adipose stem cells (ASCs) isolated from lipomatous tissue. RESULTS: We identified new biallelic LIPE null variants in three unrelated patients referred for MSL and/or partial lipodystrophy. The hallmarks of the disease, appearing in adulthood, included lower-limb lipoatrophy, upper-body and abdominal pseudo-lipomatous masses, diabetes and/or insulin resistance, hypertriglyceridemia, liver steatosis, high blood pressure, and neuromuscular manifestations. Ophthalmological investigations revealed numerous auto-fluorescent drusen-like retinal deposits in all patients. Lipomatous tissue and patient ASCs showed loss of HSL and decreased expression of adipogenic and mature adipocyte markers. LIPE-mutated ASCs displayed impaired adipocyte differentiation, decreased insulin response, defective lipolysis, and mitochondrial dysfunction. CONSLUSIONS: Biallelic LIPE null variants result in a multisystemic disease requiring multidisciplinary care. Loss of HSL expression impairs adipocyte differentiation, consistent with the lipodystrophy/MSL phenotype and associated metabolic complications. Detailed ophthalmological examination could reveal retinal damage, further pointing to the nervous tissue as an important disease target.

Platelets Facilitate the Wound-Healing Capability of Mesenchymal Stem Cells by Mitochondrial Transfer and Metabolic Reprogramming.

Platelets are known to enhance the wound-healing activity of mesenchymal stem cells (MSCs). However, the mechanism by which platelets improve the therapeutic potential of MSCs has not been elucidated. Here, we provide evidence that, upon their activation, platelets transfer respiratory-competent mitochondria to MSCs primarily via dynamin-dependent clathrin-mediated endocytosis. We found that this process enhances the therapeutic efficacy of MSCs following their engraftment in several mouse models of tissue injury, including full-thickness cutaneous wound and dystrophic skeletal muscle. By combining in vitro and in vivo experiments, we demonstrate that platelet-derived mitochondria promote the pro-angiogenic activity of MSCs via their metabolic remodeling. Notably, we show that activation of the de novo fatty acid synthesis pathway is required for increased secretion of pro-angiogenic factors by platelet-preconditioned MSCs. These results reveal a new mechanism by which platelets potentiate MSC properties and underline the importance of testing platelet mitochondria quality prior to their clinical use.

Human and murine macrophages exhibit differential metabolic responses to lipopolysaccharide - A divergent role for glycolysis.

Macrophages adopt different phenotypes in response to microenvironmental changes, which can be principally classified into inflammatory and anti-inflammatory states. Inflammatory activation of macrophages has been linked with metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis. In contrast to mouse macrophages, little information is available on the link between metabolism and inflammation in human macrophages. In the current report it is demonstrated that lipopolysaccharide (LPS)-activated human peripheral blood monocyte-derived macrophages (hMDMs) fail to undergo metabolic reprogramming towards glycolysis, but rely on oxidative phosphorylation for the generation of ATP. By contrast, activation by LPS led to an increased extracellular acidification rate (glycolysis) and decreased oxygen consumption rate (oxidative phosphorylation) in mouse bone marrow-derived macrophages (mBMDMs). Mitochondrial bioenergetics after LPS stimulation in human macrophages was unchanged, but was markedly impaired in mouse macrophages. Furthermore, treatment with 2-deoxyglucose, an inhibitor of glycolysis, led to cell death in mouse, but not in human macrophages. Finally, glycolysis appeared to be critical for LPS-mediated induction of the anti-inflammatory cytokine interleukin-10 in both human and mouse macrophages. In summary, these findings indicate that LPS-induced immunometabolism in human macrophages is different to that observed in mouse macrophages.

Platelet-Rich Plasma Improves the Wound Healing Potential of Mesenchymal Stem Cells through Paracrine and Metabolism Alterations.

Chronic and acute nonhealing wounds represent a major public health problem, and replacement of cutaneous lesions by the newly regenerated skin is challenging. Mesenchymal stem cells (MSC) and platelet-rich plasma (PRP) were separately tested in the attempt to regenerate the lost skin. However, these treatments often remained inefficient to achieve complete wound healing. Additional studies suggested that PRP could be used in combination with MSC to improve the cell therapy efficacy for tissue repair. However, systematic studies related to the effects of PRP on MSC properties and their ability to rebuild skin barrier are lacking. We evaluated in a mouse exhibiting 4 full-thickness wounds, the skin repair ability of a treatment combining human adipose-derived MSC and human PRP by comparison to treatment with saline solution, PRP alone, or MSC alone. Wound healing in these animals was measured at day 3, day 7, and day 10. In addition, we examined in vitro and in vivo whether PRP alters in MSC their proangiogenic properties, their survival, and their proliferation. We showed that PRP improved the efficacy of engrafted MSC to replace lost skin in mice by accelerating the wound healing processes and ameliorating the elasticity of the newly regenerated skin. In addition, we found that PRP treatment stimulated in vitro, in a dose-dependent manner, the proangiogenic potential of MSC through enhanced secretion of soluble factors like VEGF and SDF-1. Moreover, PRP treatment ameliorated the survival and activated the proliferation of in vitro cultured MSC and that these effects were accompanied by an alteration of the MSC energetic metabolism including oxygen consumption rate and mitochondrial ATP production. Similar observations were found in vivo following combined administration of PRP and MSC into mouse wounds. In conclusion, our study strengthens that the use of PRP in combination with MSC might be a safe alternative to aid wound healing.

MR (Mineralocorticoid Receptor) Induces Adipose Tissue Senescence and Mitochondrial Dysfunction Leading to Vascular Dysfunction in Obesity.

Adipose tissue (AT) senescence and mitochondrial dysfunction are associated with obesity. Studies in obese patients and animals demonstrate that the MR (mineralocorticoid receptor) contributes to obesity-associated cardiovascular complications through its specific role in AT. However, underlying mechanisms remain unclear. This study aims to elucidate whether MR regulates mitochondrial function in obesity, resulting in AT premature aging and vascular dysfunction. Obese (db/db) and lean (db/+) mice were treated with an MR antagonist or a specific mitochondria-targeted antioxidant. Mitochondrial and vascular functions were determined by respirometry and myography, respectively. Molecular mechanisms were probed by Western immunoblotting and real-time polymerase chain reaction in visceral AT and arteries and focused on senescence markers and redox-sensitive pathways. db/db mice displayed AT senescence with activation of the p53-p21 pathway and decreased SIRT (sirtuin) levels, as well as mitochondrial dysfunction. Furthermore, the beneficial anticontractile effects of perivascular AT were lost in db/db via ROCK (Rho kinase) activation. MR blockade prevented these effects. Thus, MR activation in obesity induces mitochondrial dysfunction and AT senescence and dysfunction, which consequently increases vascular contractility. In conclusion, our study identifies novel mechanistic insights involving MR, adipose mitochondria, and vascular function that may be of importance to develop new therapeutic strategies to limit obesity-associated cardiovascular complications.

Carbon monoxide-induced metabolic switch in adipocytes improves insulin resistance in obese mice.

Obesity is characterized by accumulation of adipose tissue and is one the most important risk factors in the development of insulin resistance. Carbon monoxide-releasing (CO-releasing) molecules (CO-RMs) have been reported to improve the metabolic profile of obese mice, but the underlying mechanism remains poorly defined. Here, we show that oral administration of CORM-401 to obese mice fed a high-fat diet (HFD) resulted in a significant reduction in body weight gain, accompanied by a marked improvement in glucose homeostasis. We further unmasked an action we believe to be novel, by which CO accumulates in visceral adipose tissue and uncouples mitochondrial respiration in adipocytes, ultimately leading to a concomitant switch toward glycolysis. This was accompanied by enhanced systemic and adipose tissue insulin sensitivity, as indicated by a lower blood glucose and increased Akt phosphorylation. Our findings indicate that the transient uncoupling activity of CO elicited by repetitive administration of CORM-401 is associated with lower weight gain and increased insulin sensitivity during HFD. Thus, prototypic compounds that release CO could be investigated for developing promising insulin-sensitizing agents.

Antioxidant properties of tea blunt ROS-dependent lipogenesis: beneficial effect on hepatic steatosis in a high fat-high sucrose diet NAFLD obese rat model.

Oxidative stress could trigger lipid accumulation in liver and thus hepatic steatosis. Tea is able to prevent liver disorders, but a direct link between antioxidant capacities and prevention of steatosis has not been reported yet. We aimed to investigate such relationship in a rat model of high fat-high sucrose diet (HFS)-induced obesity and to explore more deeply the mechanisms in isolated hepatocytes. Wistar rats were divided into a control group (standard diet), an HFS group (high fat-sucrose diet) and an HFS+tea group (HFS diet with ad-libitum access to tea drink). Body weight, fat mass, glycemic parameters in blood, lipid and oxidative stress parameters in blood and liver were measured in each group after 14 weeks. Isolated hepatocytes were treated with the reactive oxygen species (ROS) inducer t-BHP in the presence or not of antioxidants (tempol or tea), and superoxide anion production and lipid accumulation were measured using specific fluorescent probes. We reported that the HFS diet highly increased hepatic lipids content, while tea consumption attenuated steatosis and improved the oxidative status (decrease in hepatic oxidative stress, increase in plasma total antioxidant capacity). The role of antioxidant properties of tea in such phenomenon was confirmed in primary cultured rat hepatocytes. Indeed, the increase of mitochondrial ROS production with t-BHP resulted in lipid accumulation in hepatocytes (positive linear regression), and antioxidants (tempol or tea) normalized both. We reported that the antioxidant properties of tea protect rats from an obesogenic HFS diet-induced hepatic steatosis by counteracting the ROS-dependent lipogenesis.