Clusterin Neutralizes the Inflammatory and Cytotoxic Properties of Extracellular Histones in Sepsis.

Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in in vitro assays and in vivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.

Large Extracellular Vesicle-Associated Rap1 Accumulates in Atherosclerotic Plaques, Correlates With Vascular Risks and Is Involved in Atherosclerosis.

RATIONALE: Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction, but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated. OBJECTIVE: To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS. METHODS AND RESULTS: Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared with those from non-MetS subjects. Rap1 was in GTP-associated active state in both types of lEVs, and Rap1-lEVs levels correlated with increased cardiovascular risks, including stenosis. MetS-lEVs, but not non-MetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of proinflammatory molecules and activation of ERK (extracellular signal-regulated kinase) 5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 completely prevented the effects of lEVs from MetS patients. High-fat diet-fed ApoE-/- mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from high-fat diet-fed ApoE-/- mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1. CONCLUSIONS: These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, proinflammatory profile, and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis. Graphical Abstract: A graphical abstract is available for this article.

Curcumin as Prospective Anti-Aging Natural Compound: Focus on Brain.

The nutrients and their potential benefits are a new field of study in modern medicine for their positive impact on health. Curcumin, the yellow polyphenolic compound extracted from Curcuma longa species, is widely used in traditional Ayurvedic medicine to prevent and contrast many diseases, considering its antioxidant, immunomodulatory, anti-inflammatory, anti-microbial, cardio-protective, nephron-protective, hepato-protective, anti-neoplastic, and anti-rheumatic proprieties. In recent years, the investigations of curcumin have been focused on its application to aging and age-associated diseases. Aging is a physiological process in which there is a decreasing of cellular function due to internal or external stimuli. Oxidative stress is one of the most important causes of aging and age-related diseases. Moreover, many age-related disorders such as cancer, neuroinflammation, and infections are due to a low-grade chronic systemic inflammation. Curcumin acting on different proteins is able to contrast both oxidative stress than inflammation. In the brain, curcumin is able to modulate inflammation induced by microglia. Finally in brain tumors curcumin is able to reduce tumor growth by inhibition of telomerase activity. This review emphasizes the anti-aging role of curcumin focusing on its mechanism to counteract aging in the brain. Moreover, new formulations to increase the bioavailability of curcumin are discussed.

Connexin-43 is a promising target for pulmonary hypertension due to hypoxaemic lung disease.

The mechanisms underlying pulmonary hypertension (PH) are complex and multifactorial, and involve different cell types that are interconnected through gap junctional channels. Although connexin (Cx)-43 is the most abundant gap junction protein in the heart and lungs, and critically governs intercellular signalling communication, its contribution to PH remains unknown. The focus of the present study is thus to evaluate Cx43 as a potential new target in PH.Expressions of Cx37, Cx40 and Cx43 were studied in lung specimens from patients with idiopathic pulmonary arterial hypertension (IPAH) or PH associated with chronic hypoxaemic lung diseases (chronic hypoxia-induced pulmonary hypertension (CH-PH)). Heterozygous Cx43 knockdown CD1 (Cx43+/-) and wild-type littermate (Cx43+/+) mice at 12 weeks of age were randomly divided into two groups, one of which was maintained in room air and the other exposed to hypoxia (10% oxygen) for 3 weeks. We evaluated pulmonary haemodynamics, remodelling processes in cardiac tissues and pulmonary arteries (PAs), lung inflammation and PA vasoreactivity.Cx43 levels were increased in PAs from CH-PH patients and decreased in PAs from IPAH patients; however, no difference in Cx37 or Cx40 levels was noted. Upon hypoxia treatment, the Cx43+/- mice were partially protected against CH-PH when compared to Cx43+/+ mice, with reduced pulmonary arterial muscularisation and inflammatory infiltration. Interestingly, the adaptive changes in cardiac remodelling in Cx43+/- mice were not affected. PA contraction due to endothelin-1 (ET-1) was increased in Cx43+/- mice under normoxic and hypoxic conditions.Taken together, these results indicate that targeting Cx43 may have beneficial therapeutic effects in PH without affecting compensatory cardiac hypertrophy.

Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both in vitro and in vivo. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. In vitro, PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. In vivo, PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression in vivo. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis.-Bousseau, S., Marchand, M., Soleti, R., Vergori, L., Hilairet, G., Recoquillon, S., Le Mao, M., Gueguen, N., Khiati, S., Clarion, L., Bakalara, N., Martinez, M. C., Germain, S., Lenaers, G., Andriantsitohaina, R. Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Impact of polyphenols on extracellular vesicle levels and effects and their properties as tools for drug delivery for nutrition and health.

Polyphenols are found in plant-derived foods and beverages and display numerous protective effects against cancers, cardiovascular, metabolic and neurodegenerative diseases. Extracellular vesicles (EVs), microparticles, exosomes, and apoptotic bodies, originated by different cell types are emerging as a novel mean of cell-to-cell communication in physiology and pathology and represent a new way to convey fundamental information between cells. Polyphenols can act on signaling pathways that interfere with the biogenesis of EVs. Thus, they are able to control EV release from cells and their content and therefore their functional properties. Both EVs and polyphenols are therapeutic tools that can be used against several diseases. In this context, the combination of both tools can increase their therapeutic potential. Three types of strategies can be used: (i) plants are able to produce EVs that encapsulate natural components from vegetables, polyphenols for instance, (ii) mammalian cells can be treated with polyphenols and the subsequent EVs produced are enriched in these components, and (iii) EVs from mammalian cells can be uploaded with polyphenols. We review the novel aspects of the interplay between polyphenols and EVs that could trigger and improve the health benefits in cancer, cardiovascular, metabolic and neurodegenerative diseases.

Apoptotic process in cystic fibrosis cells.

Cystic fibrosis (CF) is a recessively inherited disease caused by genetic lesions in CF transmembrane conductance regulator (CFTR) gene. CF is characterized by exaggerated inflammation, progressive tissue damage, and chronic bacterial colonization, mainly in the respiratory tract. The mechanisms underlying these pathological changes are increasingly well understood. However, apoptotic dysfunction in CF disease is still debated since studies report controversial results. Nonetheless, it is clear that apoptosis participates to onset of pathology and concerns various types of cells with variable susceptibility. Apoptosis is a physiological process necessary for the preservation of homeostasis of epithelial organization and function for clearance of inflammatory cells. Increased susceptibility to apoptosis in epithelial cells and failed apoptosis in neutrophils would contribute to the self-perpetuating inflammatory cycle in CF. Also, retention of mutated CFTR in the endoplasmic reticulum participates to inflammation which may trigger apoptosis. Independently of the sensibility to apoptosis of CF cells, it has been shown that clearance of apoptotic cells, due in part to decrease in efferocytosis, is flawed and that accumulation of such cells may contribute to ongoing inflammation in CF patients. Despite great advance in understanding CF pathophysiology, there is still no cure for the disease. The most recent therapeutic strategies are directed to target CFTR protein using cell and gene therapy as well as pharmacotherapy.

Faeces-derived extracellular vesicles participate in the onset of barrier dysfunction leading to liver diseases.

The role of extracellular vesicles (EVs) from faeces (fEVs) and small circulating EVs (cEVs) in liver diseases such as non-alcoholic fatty diseases (NAFLD) and non-alcoholic steatohepatitis (NASH) has not been demonstrated. fEVs and cEVs of healthy donors, NAFLD and NASH patients were isolated and characterized. The effects of EVs were evaluated in intestinal, endothelial, Kupffer and stellate cells. Non-muscular myosin light chain kinase (nmMLCK) deficient mice were used in vivo. Bacterial origins of fEVs were analysed by 16s rDNA gene sequencing. fEVs and small cEVs were composed of prokaryotic and eukaryotic origins. Only NASH-fEVs exerted deleterious effects. NASH-fEVs increased intestinal permeability and reduced expression of tight junction proteins that were prevented by nmMLCK inhibition, increased endothelial cell permeability and inflammatory cytokines and chemokines requiring TLR4/lipopolysaccharide pathway. NASH-fEVs and NASH-cEVs activated profibrotic and proinflammatory proteins of hepatic stellate cells. Treatment with NASH-fEVs evoked an increase in intestinal permeability in wild type but not in nmMLCK deficient mice. Bacterial origins of fEVs were different between NAFLD and NASH patients and 16 amplicon sequence variants were differentially abundant. We demonstrate that fEVs actively participate in barrier dysfunctions leading to liver injuries underscoring the role of nmMLCK and lipopolysaccharide carried by fEVs.

Systems biology of antioxidants.

Understanding the role of oxidative injury will allow for therapy with agents that scavenge ROS (reactive oxygen species) and antioxidants in the management of several diseases related to free radical damage. The majority of free radicals are generated by mitochondria as a consequence of the mitochondrial cycle, whereas free radical accumulation is limited by the action of a variety of antioxidant processes that reside in every cell. In the present review, we provide an overview of the mitochondrial generation of ROS and discuss the role of ROS in the regulation of endothelial and adipocyte function. Moreover, we also discuss recent findings on the role of ROS in sepsis, cerebral ataxia and stroke. These results provide avenues for the therapeutic potential of antioxidants in a variety of diseases.

The Potential Neuroprotective Role of Free and Encapsulated Quercetin Mediated by miRNA against Neurological Diseases.

Chronic neuroinflammation is a pathological condition of numerous central nervous system (CNS) diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis and many others. Neuroinflammation is characterized by the microglia activation and concomitant production of pro-inflammatory cytokines leading to an increasing neuronal cell death. The decreased neuroinflammation could be obtained by using natural compounds, including flavonoids known to modulate the inflammatory responses. Among flavonoids, quercetin possess multiple pharmacological applications including anti-inflammatory, antitumoral, antiapoptotic and anti-thrombotic activities, widely demonstrated in both in vitro and in vivo studies. In this review, we describe the recent findings about the neuroprotective action of quercetin by acting with different mechanisms on the microglial cells of CNS. The ability of quercetin to influence microRNA expression represents an interesting skill in the regulation of inflammation, differentiation, proliferation, apoptosis and immune responses. Moreover, in order to enhance quercetin bioavailability and capacity to target the brain, we discuss an innovative drug delivery system. In summary, this review highlighted an important application of quercetin in the modulation of neuroinflammation and prevention of neurological disorders.

Age-Related Expression of IFN-λ1 Versus IFN-I and Beta-Defensins in the Nasopharynx of SARS-CoV-2-Infected Individuals.

SARS-CoV-2 coronavirus infection induces heterogeneous symptoms, ranging from asymptomatic to lethal forms. Severe forms usually occur in the elderly and/or individuals with comorbidities. Children generally remain asymptomatic to primary infection, suggesting that they may have an effective local innate immune response. IFN-I and -III have non-redundant protective roles against SARS-CoV-2, although sometimes damaging the host. The expression and role of anti-viral peptides during SARS-CoV-2 infection have thus far been little studied. We aimed to identify the innate immune molecules present at the SARS-CoV-2 entry point. We analyzed the mRNA levels of type I (IFN-α and -ß) and type III (IFN-λ1-3) interferons and selected antiviral peptides (i.e., ß-defensins 1-3, α-defensins [HNP1-3, HD5] pentraxin-3, surfactant protein D, the cathelicidin LL-37 and interleukin-26) in nasopharyngeal swabs from 226 individuals of various ages, either infected with SARS-CoV-2 (symptomatic or asymptomatic) or negative for the virus. We observed that infection induced selective upregulation of IFN-λ1 expression in pediatric subjects (≤15 years), whereas IFN-α, IFN-ß, IFN-λ2/λ3, and ß-defensin 1-3 expression was unaffected. Conversely, infection triggered upregulation of IFN-α, IFN-ß, IFN-λ2/λ3, and ß-defensin 1-3 mRNA expression in adults (15-65 years) and the elderly (≥ 65 years), but without modulation of IFN-λ1. The expression of these innate molecules was not associated with gender or symptoms. Expression of the interferon-stimulated genes IFITM1 and IFITM3 was upregulated in SARS-CoV-2-positive subjects and reached similar levels in the three age groups. Finally, age-related differences in nasopharyngeal innate immunity were also observed in SARS-CoV-2-negative subjects. This study shows that the expression patterns of IFN-I/-III and certain anti-viral molecules in the nasopharyngeal mucosa of SARS-CoV-2-infected subjects differ with age and suggests that susceptibility to SARS-CoV-2 may be related to intrinsic differences in the nature of mucosal anti-viral innate immunity.

Carrot Supplementation Improves Blood Pressure and Reduces Aortic Root Lesions in an Atherosclerosis-Prone Genetic Mouse Model.

Epidemiological studies have shown that carrot consumption may be associated with a lower risk of developing several metabolic dysfunctions. Our group previously determined that the Bolero (Bo) carrot variety exhibited vascular and hepatic tropism using cellular models of cardiometabolic diseases. The present study evaluated the potential metabolic and cardiovascular protective effect of Bo, grown under two conditions (standard and biotic stress conditions (BoBS)), in apolipoprotein E-knockout (ApoE-/-) mice fed with high fat diet (HFD). Effects on metabolic/hemodynamic parameters and on atherosclerotic lesions have been assessed. Both Bo and BoBS decreased plasma triglyceride and expression levels of genes implicated in hepatic de novo lipogenesis and lipid oxidation. BoBS supplementation decreased body weight gain, secretion of very-low-density lipoprotein, and increased cecal propionate content. Interestingly, Bo and BoBS supplementation improved hemodynamic parameters by decreasing systolic, diastolic, and mean blood pressure. Moreover, Bo improved cardiac output. Finally, Bo and BoBS substantially reduced the aortic root lesion area. These results showed that Bo and BoBS enriched diets corrected most of the metabolic and cardiovascular disorders in an atherosclerosis-prone genetic mouse model and may therefore represent an interesting nutritional approach for the prevention of cardiovascular diseases.

LPS-enriched small extracellular vesicles from metabolic syndrome patients trigger endothelial dysfunction by activation of TLR4.

BACKGROUND: Metabolic syndrome (MetS) is characterized by a cluster of interconnected risk factors -hyperglycemia, dyslipidemia, hypertension and obesity- leading to an increased risk of cardiovascular events. Small extracellular vesicles (sEVs) can be considered as new biomarkers of different pathologies, and they are involved in intercellular communication. Here, we hypothesize that sEVs are implicated in MetS-associated endothelial dysfunction. METHODS: Circulating sEVs of non-MetS (nMetS) subjects and MetS patients were isolated from plasma and characterized. Thereafter, sEV effects on endothelial function were analyzed by measuring nitric oxide (NO) and reactive oxygen species (ROS) production, and mitochondrial dynamic proteins on human endothelial aortic cells (HAoECs). RESULTS: Circulating levels of sEVs positively correlated with anthropometric and biochemical parameters including visceral obesity, glycaemia, insulinemia, and dyslipidemia. Treatment of HAoECs with sEVs from MetS patients decreased NO production through the inhibition of the endothelial NO-synthase activity. Injection of MetS-sEVs into mice impaired endothelium-dependent relaxation induced by acetylcholine. Furthermore, MetS-sEVs increased DHE and MitoSox-associated fluorescence in HAoECs, reflecting enhanced cytosolic and mitochondrial ROS production which was not associated with mitochondrial biogenesis or dynamic changes. MetS patients displayed elevated circulating levels of LPS in plasma, and, at least in part, it was associated to circulating sEVs. Pharmacological inhibition and down-regulation of TLR4, as well as sEV-carried LPS neutralization, results in a substantial decrease of ROS production induced by MetS-sEVs. CONCLUSION: These results evidence sEVs from MetS patients as potential new biomarkers for this syndrome, and TLR4 pathway activation by sEVs provides a link between the endothelial dysfunction and metabolic disturbances described in MetS.

Apple Supplementation Improves Hemodynamic Parameter and Attenuates Atherosclerosis in High-Fat Diet-Fed Apolipoprotein E-Knockout Mice.

Epidemiological studies describe the association between apple consumption and improved cardiovascular and metabolic dysfunction. Our recent multiparametric screening on cellular model studies has shown that apples exhibit vascular tropism including Granny Smith (GS) variety independently of the storage condition. The present study aimed to evaluate the cardiovascular and metabolic protection of supplementation of GS variety after storage in classic cold (GSCC) and extreme ultra-low oxygen conditions (GSXO) in the apolipoprotein E-deficient 8-week-old mice fed with high fat diet for 14 weeks. Supplementation with GSCC and GXO decreases circulating triglycerides, the expression of genes involved in lipogenesis, without change in cholesterol and glucose concentrations and HOMA-IR. Only GSXO supplementation ameliorates body weight gain, insulin level, and HDL/LDL ratio. GSXO supplementation does not modify cardiac parameters; while supplementation with GSCC decreases heart rate and improves cardiac output. Interestingly, GSCC and GSXO reduce systolic and diastolic blood pressure with a differential time course of action. These effects are associated with substantial decrease of atherosclerotic lesions. These data reinforce the knowledge about the vascular tropism of apple supplementation and underscore their ability to improve both cardiovascular and metabolic alterations in a mouse model of atherosclerosis.

Cardioprotective effect of sonic hedgehog ligand in pig models of ischemia reperfusion.

Sonic hedgehog (SHH) signaling pathway is involved in embryonic tissue patterning and development. Our previous work identified, in small rodent model of ischemia reperfusion, SHH as a specific efficient tool to reduce infarct size and subsequent arrhythmias by preventing ventricular repolarization abnormalities. The goal of the present study was to provide a proof of concept of the cardioprotective effect of SHH ligand in a porcine model of acute ischemia. Methods: The antiarrhythmic effect of SHH, either by a recombinant peptide (N-SHH) or shed membrane microparticles harboring SHH ligand (MPsSHH+), was evaluated in a first set of pigs following a short (25 min) coronary artery occlusion (CAO) followed by 24 hours-reperfusion (CAR) (Protocol A). The infarct-limiting effect was evaluated on a second set of pigs with 40 min of coronary artery occlusion followed by 24 hours reperfusion (Protocol B). Electrocardiogram (ECG) was recorded and arrhythmia's scores were evaluated. Area at risk and myocardial infarct size were quantified. Results: In protocol A, administration of N-SHH 15 min. after the onset of coronary occlusion significantly reduced the occurrence of ventricular fibrillation compared to control group. Evaluation of arrhythmic score showed that N-SHH treatment significantly reduced the overall occurrence of arrhythmias. In protocol B, massive infarction was observed in control animals. Either N-SHH or MPsSHH+ treatment reduced significantly the infarct size with a concomitant increase of salvaged area. The reduction in infarct size was both accompanied by a significant decrease in systemic biomarkers of myocardial injury, i.e., cardiac troponin I and fatty acid-binding protein and an increase of eNOS activation. Conclusions: We show for the first time in a large mammalian model that the activation of the SHH pathway by N-SHH or MPsSHH+ offers a potent protection of the heart to ischemia-reperfusion by preventing the reperfusion arrhythmias, reducing the infarct area and the circulating levels of biomarkers for myocardial injury. These data open up potentially theranostic prospects for patients suffering from myocardial infarction to prevent the occurrence of arrhythmias and reduce myocardial tissue damage.

Carrot Genotypes Contrasted by Root Color and Grown under Different Conditions Displayed Differential Pharmacological Profiles in Vascular and Metabolic Cells.

Carrots' genotype and growing conditions influence their potential properties to fight against cardiovascular and metabolic diseases. The present study evaluated the influence of carrot genotypes contrasted by root color (Bolero, Presto, Karotan, Deep Purple, Kintoki and Blanche des Vosges) growing under standard, water-restricted, biotic stress (Alternaria dauci inoculation), and combined stress conditions (water restriction and A.dauci inoculation). The effect of carrots' polyphenol and carotenoid content was assessed on endothelial and smooth muscle cells, hepatocytes, adipocytes and macrophages functions (oxidative stress, apoptosis, proliferation, lipid accumulation and inflammation). Independently of varieties or growing conditions, all carrot extracts affected vascular cells' oxidative stress and apoptosis, and metabolic cells' oxidative stress and lipid accumulation. Three clusters were revealed and displayed beneficial properties mostly for adipocytes function, smooth muscle cells and hepatocytes, and endothelial cells and hepatocytes, respectively. Karotan and Presto varieties exhibited endothelial tropism while Blanche des Vosges targeted adipocytes. Carrots under biotic stress are more efficient in inducing beneficial effects, with the Bolero variety being the most effective. However, extracts from carrots which grew under combined stress conditions had limited beneficial effects. This report underscores the use of certain carrot extracts as potential effective nutraceutical supplements for metabolic diseases.

Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors.

Microparticles (MPs) are small fragments generated from the plasma membrane after cell stimulation or apoptosis. We have recently shown that MPs harboring the morphogen Sonic Hedgehog (MPs(Shh+)) correct endothelial injury by release of nitric oxide from endothelial cells [Agouni, Mostefai, Porro, Carusio, Favre, Richard, Henrion, Martínez and Andriantsitohaina (2007) FASEB J., 21, 2735-2741]. Here, we show that MPs(Shh+) induce the formation of capillary-like structures in an in vitro model using human endothelial cells, although they inhibited cell migration. Besides, MPs(Shh+) regulate cell proliferation. Both cell adhesion and expression of proteins involved in this process such as Rho A and phosphorylation of focal-activated kinase were increased by MPs(Shh+), via a Rho-associated coiled-coil-containing protein kinase inhibitor-sensitive pathway. We demonstrate that MPs(Shh+) increase messenger RNA and protein levels of proangiogenic factors as measured by quantitative reverse transcription-polymerase chain reaction and western blot. In spite of vascular endothelial growth factor expression, conditioned media from endothelial cells treated avec MPs(Shh+) reduces angiogenesis. Interestingly, the effects induced by MPs(Shh+) on the formation of capillary-like structures, expression of adhesion molecules and proangiogenic factors were reversed after silencing of the Shh receptor, using small interfering RNA or when Sonic Hedgehog (Shh) signaling was pharmacologically inhibited with cyclopamine. Taken together, we show that Shh carried by MPs(Shh+) regulate angiogenesis probably through both a direct and an indirect mechanisms, and we propose that MPs harboring Shh may contribute to the generation of a vascular network in pathologies associated with tumor growth.

Microparticles harbouring Sonic hedgehog morphogen improve the vasculogenesis capacity of endothelial progenitor cells derived from myocardial infarction patients.

Aims: Endothelial progenitor cells (EPC) play a role in endothelium integrity maintenance and regeneration. Decreased numbers of EPC or their impaired function correlates with an increase in cardiovascular events. Thus, EPC are important predictors of cardiovascular mortality and morbidity. Microparticles carrying Sonic hedgehog (Shh) morphogen (MPShh+) trigger pro-angiogenic responses, both in endothelial cells and in ischaemic rodent models. Here, we propose that MPShh+ regulates EPC function, thus enhancing vasculogenesis, and correcting the defects in dysfunctional EPC obtained from acute myocardial infarction (AMI) patients. Methods and results: The mechanisms underlying Shh pathway function and nitric oxide (NO) production in EPC were evaluated. MPShh+ increased both the in vitro and in vivo vasculogenic capacity of EPC isolated from adult human peripheral blood samples. MPShh+ treatment significantly increased the expression of Shh signalling pathway genes (PTCH1, SMO, and GLI1) and masters of pro-angiogenic genes (NOS3, VEGFA, KDR, and KLF2) in EPC. Moreover, MPShh+ increased both the protein expression and activity of eNOS, resulting in increased NO production. Most importantly, MPShh+ improved the vasculogenic capacity of EPC from AMI patients to levels similar to that of EPC from healthy patients. All these effects were due to the activation of Shh pathway. Conclusion: MPShh+ increase both the vasculogenesis of EPC and their capacity to produce NO, including EPC from patients who have recently suffered an AMI. This study emphasizes MPShh+ and EPC as potential therapeutic tools for improving vascular regeneration as a treatment for cardiovascular ischaemic disease.

Ethanol Extract of Leaves of Cassia siamea Lam Protects against Diabetes-Induced Insulin Resistance, Hepatic, and Endothelial Dysfunctions in ob/ob Mice.

Despite long traditional utilization and some reports on the antihyperglycemic and antihyperlipidemic action of Cassia siamea, the mechanisms involved have not been investigated yet. Thus, the objective of the present study was to investigate whether and how oral administration of the ethanolic extract of Cassia siamea Lam leaves (LECS) improves glucose and insulin homoeostasis, liver damage, and endothelial dysfunction in an experimental model of type 2 diabetes, the leptin-deficient ob/ob mice. Oxidative stress and protein expression of insulin-dependent and insulin -independent signaling pathways were studied. Obese ( ob/ob) vs. control (ob/+) mice were treated daily with intragastric administration of either vehicle or LECS (200 mg/kg, per day) for 4 weeks. Fasting blood glucose, body weight, food intake, glucose and insulin tolerance, oxidative stress, and liver damage as well as vascular complications with respect to endothelial dysfunction were examined. Administration of LECS in obese mice significantly reduced blood glucose and insulin levels, improved glucose tolerance and insulin sensitivity, and restored the increase of circulating AST and ALT without modification of body weight and food intake. These effects were associated with increased activity of both insulin and AMPK pathways in the liver and skeletal muscles. Of particular interest, administration of LECS in obese mice completely prevented the endothelial dysfunction resulting from an increased NO· and decreased reactive oxygen species (ROS) production in the aorta. Altogether, oral administration of LECS remarkably attenuates features of type 2 diabetes on glucose, hepatic inflammation, insulin resistance, endothelial function, and vascular oxidative stress, being as most of these effects are related to insulin-dependent and insulin-independent mechanisms. Therefore, this study points for the therapeutic potential of Cassia siamea in correcting both metabolic and vascular alterations linked to type 2 diabetes.

Ethyl Acetate Fraction of Lannea microcarpa Engl. and K. Krause (Anacardiaceae) Trunk Barks Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice.

Traditional remedies prepared from Lannea microcarpa leaves, barks, roots, and fruits are used to treat many diseases including hypertension. This study investigated whether oral administration of the ethyl acetate fraction of Lannea microcarpa trunk barks (LMAE) corrects angiotensin (Ang) II-induced hypertension in mice. Its effects on vascular function were specifically investigated. Experiments explored hemodynamic and echocardiographic parameters in vivo and vascular reactivity to acetylcholine (ACh) and CaCl2 ex vivo on isolated aortas. Mice received LMAE for 3 weeks (50 mg/kg/day) by oral gavage. In the last two weeks of treatment, mice were implanted with osmotic minipumps delivering NaCl (0.9%) or Ang II (0.5 mg/kg/day). LMAE completely prevented the increase in systolic and diastolic blood pressure induced by Ang II. Echocardiographic and kidney parameters were not affected by the different conditions. LMAE abrogated Ang II-induced impairment of ACh-induced relaxation without affecting that of sodium nitroprusside. LMAE also completely prevented CaCl2-induced contraction in KCl-exposed aorta ex vivo. The extract alone did not modify superoxide (O2 -) and nitric oxide (NO·) production in femoral arteries from control mice but significantly limited Ang II-induced O2 - production. These effects were associated with reduced expression of inducible isoform of cyclooxygenase- (COX-) 2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform NOX-2 in aortas. Finally, phytochemical analysis showed that LMAE contains sterols, triterpenes, coumarins, and anthraquinone. These results showed that LMAE prevents Ang II-induced hypertension and vascular dysfunction through a reduction of oxidative stress linked to COX-2 and NOX-2 pathway and inhibition of calcium entry. This study provides pharmacological basis of the empirical use of Lannea microcarpa trunk bark extract against hypertension.