Neutrophils direct preexisting matrix to initiate repair in damaged tissues.

Internal organs heal injuries with new connective tissue, but the cellular and molecular events of this process remain obscure. By tagging extracellular matrix around the mesothelium lining in mouse peritoneum, liver and cecum, here we show that preexisting matrix was transferred across organs into wounds in various injury models. Using proteomics, genetic lineage-tracing and selective injury in juxtaposed organs, we found that the tissue of origin for the transferred matrix likely dictated the scarring or regeneration of the healing tissue. Single-cell RNA sequencing and genetic and chemical screens indicated that the preexisting matrix was transferred by neutrophils dependent on the HSF-integrin AM/B2-kindlin3 cascade. Pharmacologic inhibition of this axis prevented matrix transfer and the formation of peritoneal adhesions. Matrix transfer was thus an early event of wound repair and provides a therapeutic window to dampen scaring across a range of conditions.

CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

Protective effect of (Xenical+GSF) against I/R-induced blood brain barrier disruption, ionic edema, lipid deregulation and neuroinflammation.

Ischemic stroke is a leading cause of mortality worldwide that occurs following the reduction or interruption of blood brain supply, characterized by a cascade of early events as oxidative stress and ensuing neuro-inflammation, energy failure and the burst of intracellular Ca++ resulting in activation of phospholipases and large increase in FFA including arachidonic acid, ultimately leading to nervous cell death. Grape Seed Flour (GSF) is a complex polyphenolic mixture harboring antioxidant, anti-inflammatory and neuroprotective properties. Orlistat (Xenical ™,Xe) is a gastro-intestinal lipase inhibitor and an anti-obesity agent. In an earlier study we reported the higher efficiency in neuroprotection against HFD-induced brain lipotoxicity when combining the two drugs (GSF + Xe). As a result repurposing Xe as an adjunct to GSF therapy against stroke appeared relevant and worthy of investigation. I/R insult disrupted the blood brain barrier (BBB) as assessed by EB dye extravasation, increased water and Na+ within the brain. Ultrastructurally I/R altered the brain blood capillaries at the vicinity of hippocampus dentate gyrus area as assessed by transmission and scanning electron microscopy. I/R altered lipid metabolism as revealed by LDL/HDL ratio, lipase activity, and FFA profiles. Moreover, I/R induced neuro-inflammation as assessed by down-regulation of anti-inflammatory CD 56 and up-regulation of pro-inflammatory CD 68 antigen. Importantly almost all I/R-induced disturbances were retrieved partially upon Xe or GSF on their own, and optimally when combining the two drugs. Xe per se is protective against I/R injury and the best neuroprotection was obtained when associating low dosage Xe with high dosage GSF, enabling neuroprevention and cell survival within hippocampus dentate gyrus area as revealed by increased staining of Ki 67 proliferation biomarker.

Alleviation of drought stress in faba bean (Vicia faba L.) by exogenous application of ß-aminobutyric acid (BABA).

Drought stress is one of the most prevalent environmental factors limiting faba bean (Vicia faba L.) crop productivity. ß-aminobutyric acid (BABA) is a non-protein amino acid that may be involved in the regulation of plant adaptation to drought stress. The effect of exogenous BABA application on physiological, biochemical and molecular responses of faba bean plants grown under 18% PEG-induced drought stress were investigated. The results showed that the application of 1 mM of BABA improved the drought tolerance of faba bean. The application of BABA increased the leaf relative water content, leaf photosynthesis rate (A), transpiration rate (E), and stomatal conductance (gs), thereby decreased the water use efficiency. Furthermore, exogenous application of BABA decreased production of hydrogen peroxide (H2O2), malondialdehyde and electrolyte leakage levels, leading to less cell membrane damage due to oxidative stress. Regarding osmoprotectants, BABA application enhanced the accumulation of proline, and soluble sugars, which could improve the osmotic adjustment ability of faba bean under drought challenge. Interestingly, mended antioxidant enzyme activities like catalase, guaiacol peroxidase, ascorbate peroxidase and superoxide dismutase and their transcript levels may lead to counteract the damaging effects of oxidative stress and reducing the accumulation of harmful substances in BABA-treated faba bean plants. In addition, exogenous BABA significantly induced the accumulation of drought tolerance-related genes like VfMYB, VfDHN, VfLEA, VfERF, VfNCED, VfWRKY, VfHSP and VfNAC in leaves and roots, suggesting that BABA might act as a signal molecule to regulate the expression of drought tolerance-related genes.

Neuroprotective effect of grape seed extract on brain ischemia: a proteomic approach.

Stroke is one of the leading causes of long-lasting disability in human and oxidative stress an important underlying cause. Molecular insights into pathophysiology of ischemic stroke are still obscure, and the present study investigated the protective effect of high dosage Grape Seed Extract (GSE 2.5 g/kg) on brain ischemia-reperfusion (I/R) injury using a proteomic approach. Ischemia was realized by occlusion of the common carotid arteries for 30 min followed by 1 h reperfusion on control or GSE pre-treated rats, and a label-free quantification followed by mass spectrometry analysis used to evaluate I/R induced alterations in protein abundance and metabolic pathways as well as the protection afforded by GSE. I/R-induced whole brain ionogram dyshomeostasis, ultrastructural alterations, as well as inflammation into hippocampal dentate gyrus area, which were evaluated using ICP-OES, transmission electron microscopy and immuno-histochemistry respectively. I/R altered the whole brain proteome abundance among which 108 proteins were significantly modified (35 up and 73 down-regulated proteins). Eighty-four proteins were protected upon GSE treatment among which 27 were up and 57 down-regulated proteins, suggesting a potent protective effect of GSE close to 78%of the disturbed proteome. Furthermore, GSE efficiently prevented the brain from I/R-induced ion dyshomeostasis, ultrastructural alterations, inflammatory biomarkers as CD56 or CD68 and calcium burst within the hippocampus. To conclude, a potent protective effect of GSE on brain ischemia is evidenced and clinical trials using high dosage GSE should be envisaged on people at high risk for stroke.

Protective effect of grape seed and skin extract against high-fat diet-induced dyshomeostasis of energetic metabolism in rat lung.

BACKGROUND: Obesity is currently one of the major epidemics of this millennium and affects poeples throughout the world. It causes multiple systemic complications as it significantly interferes with respiratory function. OBJECTIVE: We aimed in the present work to study the effect of high fat diet (HFD) on lung oxidative stress and energy metabolism alterations, as well as the putative protection afforded by grape seed and skin extract (GSSE). METHODS: We started by characterizing the GSSE and its composition using gas chromatography coupled to mass spectrometry (GC-MS). We used a rat model of high-fat-diet and we evaluated the effect of GSSE on oxidative stress and energetic disturbances induced by HFD. We analyzed the effect of HFD on lung oxidative status by assessing lipid oxidation level, non-protein thiols (NPSH) and superoxide anion level… We also evaluated the effect of HFD on creatine kinase (CK), malate dehydrogenase (MDH) and mitochondrial complex IV. RESULTS: HFD induced body weight gain, increased lung weight and lipid content without affecting insulinemia and dropped adiponectemia. HFD also provoked on lung oxidative stress characterized by increased carbonylation (+ 95%; p = 0.0045), decreased of NPSH (- 32%; p = 0.0291) and inhibition of antioxidant enzyme activities such as glutathione peroxidase (- 25%; p = 0.0074). HFD also altered lung intracellular mediators as superoxide anion O2¯ (+ 59%; p = 0.0027) and increased lung xanthine oxidase activity (+ 27%; p = 0.0122). HFD induced copper depletion (- 24%; p = 0.0498) and lead (- 51%: p = 0.0490) from the lung. Correlatively HFD decreased the copper associated enzyme tyrosinase (- 29%; p = 0.0500) and decreased glutamine synthetase activity (- 31%; p = 0.0027). HFD altered also lung energy metabolism by increasing CK activity (+ 22%; p = 0.0108) and decreasing MDH and mitochondrial complex IV activities (- 28%; p = 0.0120, - 31%; p = 0.0086 respectively). Importantly all these alterations were efficiently corrected with GSSE treatment. CONCLUSION: In conclusion, GSSE has the potential to alleviate the deleterious lipotoxic effect of HFD on lung and it could find potential application in the protection against HFD-induced lung complications.

Brain proteomic modifications associated to protective effect of grape extract in a murine model of obesity.

In recent years, the obesity epidemic has developed into a major health crisis worldwide. With current treatments limited to expensive, high-risk surgery and minimally efficacious pharmacotherapy, new therapeutic options are urgently needed to fight against this alarming trend. Though brain dysfunction has been studied linked to high fat diet (HFD) and grape seed and skin extract (GSSE) correction, the proteomic modifications linking the two effects on brain lipotoxicity are not well understood. To this end rats were exposed for 8 weeks to HFD treatment, to GSSE (500mg/kg BW) and to binary mixture of HFD and GSSE to gain insight into the potential pathways altered with metabolic disease and the protection afforded by GSSE. Significant modifications of brain proteins were detected using mass spectrometry-based differential proteomics. These proteins were mainly related to oxidative stress, glycolysis and calcium signaling. Additionally, proteins involved in the cytoskeleton were also affected by HFD treatment. Interestingly, whether up- or down regulated protein abundances, GSSE corrected most of the disturbances of HFD treatment. These findings provide impetus for future therapeutic investigation on GSSE against other metabolic disorders.

Protective effects of grape seed and skin extract against high-fat-diet-induced lipotoxicity in rat lung.

BACKGROUND: Obesity is a public health problem characterized by increased fat accumulation in different tissues. Obesity is directly linked to breathing problems and medical complications with lung, including obstructive sleep apnea syndrome, obesity hypoventilation syndrome, chronic obstructive pulmonary disease, asthma….In the present work, we aimed to investigate the effect of high fat diet (HFD) on lung lipotoxicity, oxidative stress, fatty acid composition and proportions in lung and implication in asthma development. The likely protection provided by grape seed extract (GSSE) was also investigated. METHODS: In order to assess HFD effect on lung and GSSE protection we used a rat model. We analyzed the lipid plasma profile, lung peroxidation and antioxidant activities (SOD, CAT and POD). We also analyzed transition metals (Ca2+, Mg2+, Zn2+ and iron) and lung free fatty acids using gas chromatography coupled to mass spectrometry (GC-MS). RESULTS: HFD induced lipid profile imbalance increasing cholesterol and VLDL-C. HFD also induced an oxidative stress assessed by elevated MDA level and the drop of antioxidant activities such as SOD, CAT and POD. Moreover, HFD induced mineral disturbances by decreasing magnesium level and increasing Calcium and iron levels. HFD induced also disturbances in lung fatty acid composition by increasing oleic, stearic and arachidonic acids. Interestingly, GSSE alleviated all these deleterious effects of HFD treatment. CONCLUSION: As a whole, GSSE had a significant preventive effect against HFD-induced obesity, and hence may be used as an anti-obesity agent, and a benefic agent with potential applications against damages in lung tissue.

Grape seed and skin extract protects kidney from doxorubicin-induced oxidative injury.

The study investigated the protective effect of grape seed and skin extract (GSSE) against doxorubicin-induced renal toxicity in healthy rats. Animals were treated with GSSE or not (control), for 8 days, administered with doxorubicin (20mg/kg) in the 4th day, and renal function as well as oxidative stress parameters were evaluated. Data showed that doxorubicin induced renal toxicity by affecting renal architecture and plasma creatinine. Doxorubicin also induced an oxidative stress characterized by an increase in malondialdehyde (MDA), calcium and H(2)O(2) and a decrease in catalase (CAT) and superoxide dismutase (SOD). Unexpectedly doxorubicin increased peroxidase (POD) and decreased carbonyl protein and plasma urea. Treatment with GSSE counteracted almost all adverse effects induced by doxorubicin. Data suggest that doxorubicin induced an oxidative stress into rat kidney and GSSE exerted antioxidant properties, which seem to be mediated by the modulation of intracellular calcium.

Impact of Curcumin on the IL-17A-Mediated p53-Fibrinolytic System: Mouse Proteomics and Integrated Human Fibrosis scRNAseq Insights.

Acute lung injury (ALI) is primarily driven by an intense inflammation in the alveolar epithelium. Key to this is the pro-inflammatory cytokine, Interleukin 17 (IL-17), which influences pulmonary immunity and modifies p53 function. The direct role of IL-17A in p53-fibrinolytic system is still unclear, it is important to evaluate this mechanism to regulate the ALI progression to idiopathic pulmonary fibrosis (IPF). C57BL/6 mice, exposed to recombinant IL-17A protein and treated with curcumin, provided insight into IL-17A mechanisms and curcumin's potential for modulating early pulmonary fibrosis stages. A diverse methodology, including proteomics, single-cell RNA sequencing (scRNA-seq) integration, molecular, and Schroedinger approach were utilized. In silico approaches facilitated the potential interactions between curcumin, IL-17A, and apoptosis-related proteins. A notable surge in the expression levels of IL-17A, p53, and fibrinolytic components such as Plasminogen Activator Inhibitor-1 (PAI-I) was discerned upon the IL17A exposure in mouse lungs. Furthermore, the enrichment of pathways and differential expression of proteins underscored the significance of IL-17A in governing downstream regulatory pathways such as inflammation, NF-kappaB signaling, Mitogen-Activated Protein Kinases (MAPK), p53, oxidative phosphorylation, JAK-STAT, and apoptosis. The integration of scRNA-seq data from 20 IPF and 10 control lung specimens emphasized the importance of IL-17A mediated downstream regulation in PF patients. A potent immuno-pharmacotherapeutic agent, curcumin, demonstrated a substantial capacity to modulate the lung pathology and molecular changes induced by IL-17A in mouse lungs. Human IPF single cell data integration confirmed the effects of IL-17A mediated fibrinolytic components in ALI to IPF progression.

RNA-Seq of Single Fish Cells - Seeking Out the Leukocytes Mediating Immunity in Teleost Fishes.

The immune system is a complex and sophisticated biological system, spanning multiple levels of complexity, from the molecular level to that of tissue. Our current understanding of its function and complexity, of the heterogeneity of leukocytes, is a result of decades of concentrated efforts to delineate cellular markers using conventional methods of antibody screening and antigen identification. In mammalian models, this led to in-depth understanding of individual leukocyte subsets, their phenotypes, and their roles in health and disease. The field was further propelled forward by the development of single-cell (sc) RNA-seq technologies, offering an even broader and more integrated view of how cells work together to generate a particular response. Consequently, the adoption of scRNA-seq revealed the unexpected plasticity and heterogeneity of leukocyte populations and shifted several long-standing paradigms of immunology. This review article highlights the unprecedented opportunities offered by scRNA-seq technology to unveil the individual contributions of leukocyte subsets and their crosstalk in generating the overall immune responses in bony fishes. Single-cell transcriptomics allow identifying unseen relationships, and formulating novel hypotheses tailored for teleost species, without the need to rely on the limited number of fish-specific antibodies and pre-selected markers. Several recent studies on single-cell transcriptomes of fish have already identified previously unnoticed expression signatures and provided astonishing insights into the diversity of teleost leukocytes and the evolution of vertebrate immunity. Without a doubt, scRNA-seq in tandem with bioinformatics tools and state-of-the-art methods, will facilitate studying the teleost immune system by not only defining key markers, but also teaching us about lymphoid tissue organization, development/differentiation, cell-cell interactions, antigen receptor repertoires, states of health and disease, all across time and space in fishes. These advances will invite more researchers to develop the tools necessary to explore the immunology of fishes, which remain non-conventional animal models from which we have much to learn.

Grape Seed Flour Extends Longevity by Improving Multi-Organ Dysfunction and Age-Associated Oxidative Stress and Inflammation in Healthy Rat.

According to the free-radical theory of aging, accumulation of reactive oxygen species (ROS) within mitochondria throughout life span leads to impairment of the main biological macromolecules as DNA, lipids, and proteins, which might be at the basis of premature aging. One way to test experimentally such a hypothesis consists in intervention studies using antioxidant nutrients aimed at limiting or inhibiting ROS production that should be able to reduce the aging rate and disease pathogenesis. Grape seed flour (GSF) contains a high level of phytochemicals among which bioactive polyphenols exhibit numerous biological properties and beneficial health effects as antioxidant, anti-inflammatory, anticarcinogenic, multi-organ (heart, liver, kidney, and brain among others) protective. The present study aimed at testing the ability of high dosing GSF (4 g/kg bw) used as a nutritional supplement to slow down aging and prolong life span of Wistar rats when administered from early life (1-month-old animals) till their natural death. Data clearly show that high-dose GSF extends organism longevity and health span by improving multi-organ damages, systemic fueling metabolism declines, and alleviated oxidative stress and inflammation in aging rats. Our data support the extending longevity effect of grape polyphenols especially when used as high dosing nutritional supplement or as natural medicine whose appropriate galenic form as solid lipid nanoformulation is currently under investigation.

Protective effect of grape seed extract and orlistat co-treatment against stroke: Effect on oxidative stress and energy failure.

Ischemic stroke is a major health concern and a leading cause of mortality worldwide. Oxidative stress is an early event in the course of stroke inducing neuro-inflammation and cell death. Grape seed extract (GSE) is a natural phytochemical mixture exhibiting antioxidant, anti-inflammatory and neuroprotective properties. Orlistat (ORL) is an anti-obesity agent and a gastro-intestinal lipase inhibitor which showed recently beneficial effects on brain lipotoxicity. Recent studies reported the increase of lipase activity upon stroke which led us to investigate the neuroprotective effect of ORL on rat brain I/R injury as well as the putative synergism with GSE. I/R insult infarcted the brain parenchyma as assessed by TTC staining, induced an oxidative stress as revealed by increased lipoperoxidation along with alteration of antioxidant enzymes activities which was corrected using the cotreatment of ORL + GSE. I/R also disturbed the main metabolic pathways involved in brain fueling as glycolysis, neoglucogenesis, glycogenolysis, TCA cycle and electron transfer chain (ETC) complexes. These disturbances were also corrected with the cotreatment ORL + GSE which maintained energetic activities near to the control level. I/R also disrupted transition metals distribution, along with associated enzymes as tyrosinase, LDH or glutamine synthetase activities and induced hippocampal inflammation as revealed by glycogen depletion from dentate gyrus area along with depressed anti-inflammatory IL1ß cytokine and increased pro-inflammatory CD68 antigen. Interestingly almost all I/R-induced disturbances were corrected either partially upon ORL and GSE on their own and the best neuroprotection was obtained in the presence of both drugs (ORL + GSE) enabling robust neuroprotection of the sub granular zone within hippocampal dentate gyrus area.

Preventive and curative effects of grape seed powder on stroke using in vitro and in vivo models of cerebral ischemia/reperfusion.

Stroke is a worldwide concern. Many studies pointed out relevant preventive effect of grape seed powder (GSP) against deleterious brain ischemia/reperfusion (I/R) injury, but curative effect has been scarcely approached. The present work aimed at studying the preventive and curative effect of GSP against stroke using in-vitro and in-vivo models. Primary neuron-astrocyte cocultures were used to evaluate in-vitro GSP protective and curative effect on oxygen-glucose-deprivation (OGD). A murine I/R model, in which GSP was administered as delayed post stroke drug, to evaluate its potential clinically translatable therapy was used and behavioral tests were conducted after 15 days. Ultra-structure of hippocampus dentate gyrus using Transmission Electron Microscopy (TEM) was also undertaken. GSP prevented OGD-induced toxicity and cell death in a dose dependent manner and was neuroprotective as assessed by sustained cell viability (70 % ±1 for OGD + GSP and 37 % ±2 for OGD) and modulated cytokines and brain derived neurotrophic factor (BDNF) expression. GSP also promoted behavioral outcomes by increasing step-down inhibitory time from 17s±4 to 50s±11 and rat overall activities by improving scores in open field test to near control level. Furthermore, GSP protected hippocampus dentate gyrus area from I/R-induced drastic alterations as assessed by reduced autophagic vacuoles.

Characterization, anti-oxidative effect of grape seed powder and in silico affinity profiling of polyphenolic and extra-phenolic compounds for calpain inhibition.

Vitis vinifera grape is a highly cultivated crop and solid wastes generated by the wine industry are largely under exploited. Plentiful studies have intended analyzing the polyphenolic content of grape seeds but characterization of non phenolic compounds is rather scarce. The present study aimed at the selective extraction of lipid, phenolic and aqueous phases from grape seed powder (GSP) in order to establish their intimate composition, as well as their antioxidant and chelating properties underlying partly their biological effects. Major non phenolic compounds identified in the lipid phase were glyceryl-monostearate and 2-monostearin whereas fructofuranose and sucrose were the most abundant in the aqueous phase. Among the most abundant compounds detected in the various phases, the polyphenol quercetin exhibited the best affinity and free binding energy towards the active site of the calcium-dependent protease calpain. Polyphenols likely constitute the bioactive part of GSP that should be exploited as safe modulators of intracellular signaling which is likely at the basis of their health beneficial effects. Nevertheless other compounds as lipids or sugars should be valorized along with polyphenols to improve their bioavailability into highly protected organs as brain or eye.

Dietary supplementation of grape seed and skin flour mitigates brain oxidative damage induced by a high-fat diet in rat: Gender dependency.

It is unknown whether gender has an impact on brain injury in obesity, and, if so, whether treatment with grape seed and skin flour could exert a protective effect. Both male and female rats were fed a standard diet (SD) or a high fat diet (HFD) during eight weeks and treated with high dosage grape seed and skin flour (GSSF). Fat-induced oxidative stress was evaluated into the brain with a special emphasis on transition metals determination. HFD induced male-cholesterol overload (+78.12%) and an oxidative stress status characterized by increased lipoperoxidation (+68.97%), carbonylation (+40.28%), decreased antioxidant enzyme activities as glutathione peroxidase (-61.07%) and manganese-superoxide dismutase (-35.47%) but not catalase. Additionally HFD depleted the brain from manganese (-71.31%) and dropped glutamine synthetase activity (-36.16%), without affecting copper nor iron nor their associated enzymes. HFD also altered intracellular mediators as superoxide anion (+36.12%), calcium (+44.41%) and also calpain (+76.54%) a calcium dependent protease. Importantly all these alterations were detected exclusively in male brain and were efficiently corrected upon GSSF treatment. In conclusion, GSSF has the potential to alleviate the deleterious lipotoxic effect of HFD treatment that occurred in male brain and perhaps in post-menauposal female brain.