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.

Lipidomic analysis of adipose-derived extracellular vesicles reveals specific EV lipid sorting informative of the obesity metabolic state.

Adipose extracellular vesicles (AdEVs) transport lipids that could participate in the development of obesity-related metabolic dysfunctions. This study aims to define mouse AdEV lipid signature by a targeted LC-MS/MS approach in either healthy or obesity context. Distinct clustering of AdEV and visceral adipose tissue (VAT) lipidomes by principal component analysis reveals specific AdEV lipid sorting when compared with secreting VAT. Comprehensive analysis identifies enrichment of ceramides, sphingomyelins, and phosphatidylglycerols species in AdEVs compared with source VAT whose lipid content closely relates to the obesity status and is influenced by the diet. Obesity moreover impacts AdEV lipidome, mirroring lipid alterations retrieved in plasma and VAT. Overall, our study identifies specific lipid fingerprints for plasma, VAT, and AdEVs that are informative of the metabolic status. Lipid species enriched in AdEVs in the obesity context may constitute biomarker candidates or mediators of the obesity-associated metabolic dysfunctions.

Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

Recent evidence supporting that adipose tissue (AT)-derived extracellular vesicles (EVs) carry an important part of the AT secretome led us to characterize the EV-adipokine profile. In addition to evidencing a high AT-derived EV secretion ability that is further increased by obesity, we identify enrichment of oligomeric forms of adiponectin in small EVs (sEVs). This adipokine is mainly distributed at the EV external surface as a result of nonspecific adsorption of soluble adiponectin. EVs also constitute stable conveyors of adiponectin in the blood circulation. Adiponectin-enriched sEVs display in vitro insulin-sensitizing effects by binding to regular adiponectin receptors. Adoptive transfer of adiponectin-enriched sEVs in high-fat-diet-fed mice prevents animals from gaining weight and ameliorated insulin resistance and tissue inflammation, with major effects observed in the AT and liver. Our results therefore provide information regarding adiponectin-related metabolic responses by highlighting EVs as delivery platforms of metabolically active forms of adiponectin molecules.

Pharmacokinetics of intact lipid nanocapsules using new quantitative FRET technique.

The present study investigated the pharmacokinetics of intact lipid nanocapsules (LNCs) after intravenous administration in rats. Six different Förster resonance energy transfer LNCs (FRET-LNCs) have been studied with 2 sizes (50 and 85 nm) and 3 coating types (none, DSPE-mPEG 2000 or stearylamine). A FRET-LNCs blood extraction method was developed to retain an accurate FRET signal. Intact FRET-LNCs were specifically quantified through combination of FRET signal and Nano Tracker Analysis. Pharmacokinetic data were first described by non-compartmental analysis, then used to develop a population pharmacokinetic model. The pharmacokinetic elimination of FRET-LNCs was non-linear and dependent on size and surface modification, while the distribution was dependent on size. The LNCs 85 nm volume of distribution was lower than LNCs 50 nm. As expected, LNCs 85 nm with PEG coating displayed a lower clearance than other formulations. Surprisingly, this study highlighted a faster elimination of LNCs 50 nm with PEG compared to other formulations which could be explained by instability in blood. This first pharmacokinetic model of intact LNCs allowed a thorough understanding of the influence of size and coating on pharmacokinetic properties and paves the way for future mechanistic modeling approaches to predict the fate of LNCs in vivo.

Pharmacology active microcarriers delivering HGF associated with extracellular vesicles for myocardial repair.

Despite the curative approaches developed against myocardial infarction, cardiac cell death causes dysfunctional heart contractions that depend on the extent of the ischemic area and the reperfusion period. Cardiac regeneration may allow neovascularization and limit the ventricular remodeling caused by the scar tissue. We have previously found that large extracellular vesicles, carrying Sonic Hedgehog (lEVs), displayed proangiogenic and antioxidant properties, and decreased myocardial infarction size when administrated by intravenous injection. We propose to associate lEVs with pharmacology active microcarriers (PAMs) to obtain a combined cardioprotective and regenerative action when administrated by intracardiac injection. PAMs made of poly-D,L-lactic-coglycolic acid-poloxamer 188-poly-D,L-lactic-coglycolic acid and covered by fibronectin/poly-D-lysine provided a biodegradable and biocompatible 3D biomimetic support for the lEVs. When compared with lEVs alone, lEVs-PAMs constructs possessed an enhanced in vitro pro-angiogenic ability. PAMs were designed to continuously release encapsulated hepatocyte growth factor (PAMsHGF) and thus, locally increase the activity of the lEVs by the combined anti-fibrotic properties and regenerative properties. Intracardiac administration of either lEVs alone or lEVs-PAMsHGF improved cardiac function in a similar manner, in a rat model of ischemia-reperfusion. Moreover, lEVs alone or the IEVs-PAMsHGF induced arteriogenesis, but only the latter reduced tissue fibrosis. Taken together, these results highlight a promising approach for lEVs-PAMsHGF in regenerative medicine for myocardial infarction.

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.

Small extracellular vesicle-mediated targeting of hypothalamic AMPKα1 corrects obesity through BAT activation.

Current pharmacological therapies for treating obesity are of limited efficacy. Genetic ablation or loss of function of AMP-activated protein kinase alpha 1 (AMPKα1) in steroidogenic factor 1 (SF1) neurons of the ventromedial nucleus of the hypothalamus (VMH) induces feeding-independent resistance to obesity due to sympathetic activation of brown adipose tissue (BAT) thermogenesis. Here, we show that body weight of obese mice can be reduced by intravenous injection of small extracellular vesicles (sEVs) delivering a plasmid encoding an AMPKα1 dominant negative mutant (AMPKα1-DN) targeted to VMH-SF1 neurons. The beneficial effect of SF1-AMPKα1-DN-loaded sEVs is feeding-independent and involves sympathetic nerve activation and increased UCP1-dependent thermogenesis in BAT. Our results underscore the potential of sEVs to specifically target AMPK in hypothalamic neurons and introduce a broader strategy to manipulate body weight and reduce obesity.

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.

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.

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.

Screening of ordinary commercial varieties of apple fruits under different storage conditions for their potential vascular and metabolic protective properties.

Epidemiological studies reported that apple consumption is associated with a decrease of cardiovascular and metabolic dysfunction, probably due to the polyphenols and fibers present in this fruit. The storage conditions and genetic origin of apples have been reported to influence their content and, as a consequence, their pharmacological properties. The present study evaluated the influence of varieties and storage conditions of traditional and highly appreciated apples including Gala, Golden Delicious, Granny Smith and Pink Lady varieties after harvest and storage under classic cold conditions, under a controlled atmosphere, or under extreme ultra-low oxygen conditions. Thus, a multi-parametric screening on cell models associated with vascular and metabolic dysfunctions - such as endothelial and smooth muscle cells, hepatocytes, adipocytes and macrophages - in relation to the apple polyphenol content has been developed. This strategy demonstrated that, overall, peeled apple samples exhibited a vascular tropism and acted mainly on proliferation and oxidative stress in endothelial and smooth muscle cells. Apple extracts appeared to be less effective on adipocytes and macrophages, but they exhibited antioxidant properties in hepatocytes. Among the varieties, Gala and Golden Delicious were the most efficient against the processes involved in the development of atherosclerosis. Concerning storage conditions, most of the apple varieties were more efficient under harvest conditions, while they could not be discriminated under all other cold conditions and the concentration used, except for the Gala samples. Interestingly, pharmacological properties were associated with the polyphenol profiles of freeze dried apple flesh powder. The present report revealed the potential use of some apple extracts as effective food supplements or nutraceuticals for the prevention and/or management of cardiovascular and metabolic diseases.

Estrogen receptor α/HDAC/NFAT axis for delphinidin effects on proliferation and differentiation of T lymphocytes from patients with cardiovascular risks.

Delphinidin, an anthocyanin present in red wine, has been reported to preserve the integrity of endothelium via an estrogen receptor alpha (ERα)-dependent mechanism. However, the effect of delphinidin on the immune response in obesity-related inflammation remains unknown. Given the important role of T lymphocytes in obesity-related inflammation, we investigated the effect of delphinidin on proliferation and differentiation of T lymphocytes from healthy subjects and metabolic syndrome patients. Delphinidin decreased the proliferation stimulated by different agents acting through different mechanisms. This effect of delphinidin was associated with its ability to inhibit Ca2+ signaling via reduced store-operated Ca2+ entry and release, and subsequent decrease of HDAC and NFAT activations. Delphinidin also inhibited ERK1/2 activation. Pharmacological inhibition of ER with fulvestrant, or deletion of ERα, prevented the effect of delphinidin. Further, delphinidin suppressed the differentiation of T cells toward Th1, Th17 and Treg without affecting Th2 subsets. Interestingly, delphinidin inhibited both proliferation and differentiation of T cells taken from patients with cardiovascular risks associated with metabolic syndrome. Together, we propose that delphinidin, by acting on ERα via multiple cellular targets, may represent a new approach against chronic inflammation associated with T lymphocyte activation, proliferation and differentiation, in patients with cardiovascular risk factors.

Characterisation of adipocyte-derived extracellular vesicle subtypes identifies distinct protein and lipid signatures for large and small extracellular vesicles.

Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

Microparticles from apoptotic RAW 264.7 macrophage cells carry tumour necrosis factor-α functionally active on cardiomyocytes from adult mice.

After ischaemic injury and in patients with atherosclerosis, the pool of inflammatory macrophages is enlarged in the heart and in atherosclerotic plaques. Monocyte/macrophage-derived microparticles (MPs) are part of the pathological process of unstable atherosclerotic plaques. The present study focused on effects of MPs, produced by apoptotic murine RAW 264.7 macrophage cell line, in adult murine cardiomyocytes. Flow cytometry and western blot analysis showed that these MPs contained the soluble form of tumour necrosis factor alpha (TNF-α). Cardiomyocyte sarcomere shortening amplitudes and kinetics were reduced within 5 min of exposure to these MPs. Conversely, Ca(2+) transient amplitude and kinetics were not modified. The contractile effects of MPs were completely prevented after pretreatment with nitric oxide synthase, guanylate cyclase or TNF-α inhibitors as well as blocking TNF-α receptor 1 with neutralizing antibody. Microscopy showed that, after 1 h, MPs were clearly surrounding rod-shaped cardiomyocytes, and after 2 h they were internalized into cardiomyocytes undergoing apoptosis. After 4 h of treatment with MPs, cardiomyocytes expressed increased caspase-3, caspase-8, Bax and cytochrome C. Thus, MPs from apoptotic macrophages induced a negative inotropic effect and slowing of both contraction and relaxation, similar to that observed in the presence of TNF-α. The use of specific inhibitors strongly suggests that TNF-α receptors and the guanylate cyclase/cGMP/PKG pathway were involved in the functional responses to these MPs and that the mitochondrial intrinsic pathway was implicated in their proapoptotic effects. These data suggest that MPs issued from activated macrophages carrying TNF-α could contribute to propagation of inflammatory signals leading to myocardial infarction.