Recombinant human activated protein C improves endotoxemia-induced endothelial dysfunction: a blood-free model in isolated mouse arteries

Recombinant human activated protein C (rhAPC) is one of the treatment panels for improving vascular dysfunction in septic patients. In a previous study, we reported that rhAPC treatment in rat endotoxemia improved vascular reactivity, although the mechanisms involved are still under debate. In the present study, we hypothesized that rhAPC may improve arterial dysfunction through its nonanticoagulant properties. Ten hours after injection of LPS in mice (50 mg/kg ip), aortic rings and mesenteric arteries were isolated and incubated with or without rhAPC for 12 h. Aortic rings were mounted in a myograph, after which arterial contractility and endothelium-dependent relaxation were measured in the presence or absence of nitric oxide synthase or cyclooxygenase inhibitors. Flow (shear stress)-mediated dilation with or without the above inhibitors was also measured in mesenteric resistance arteries. Protein expression was assessed by Western blotting. Lipopolysaccharide (LPS) reduced aortic contractility to KCl and phenylephrine as well as dilation to acetylcholine. LPS also reduced flow-mediated dilation in mesenteric arteries. In rhAPC-treated aorta and mesenteric arteries, contractility and endothelial responsiveness to vasodilator drug and shear stress were improved. rhAPC treatment also improved LPS-induced endothelial dysfunction; this effect was associated with an increase in the phosphorylated form of endothelial nitric oxide synthase and protein kinase B as well as cyclooxygenase vasodilatory pathways, thus suggesting that these pathways, together with the decrease in nuclear factor-κB activation and inducible nitric oxide synthase expression in the vascular wall, are implicated in the endothelial effect of rhAPC. In conclusion, ex vivo application of rhAPC improves arterial contractility and endothelial dysfunction resulting from endotoxemia in mice. This finding provides important insights into the mechanism underlying rhAPC-induced improvements on arterial dysfunction during septic shock

Red wine polyphenols prevent metabolic and cardiovascular alterations associated with obesity in Zucker fatty rats (Fa/Fa)

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Human serum albumin improves endothelial dysfunction and survival during experimental endotoxemia: Concentration-dependent properties

Objectives: The mechanisms by which human serum albumin might protect against sepsis-induced organ dysfunction and improve survival are not elucidated. The present study was designed to assess the effects of two concentrations of human serum albumin on endotoxin-induced mortality as well as on endothelial and organ dysfunctions in both mouse and cell models.Design: Prospective, randomized, controlled experimental study. Setting: University research laboratories. Subjects: Swiss mice (n = 10–15/group) were injected with either lipopolysaccharide or vehicle. Four and 12 hrs later, mice were infused or not with human serum albumin HSA (4% or 20%, 10 mL/kg) or normal saline (0.9% NaCl, 30 mL/kg). Human uterine vein endothelial cells were exposed to both lipopolysaccharide and tumor necrosis factor-α during 8 hrs in the presence or absence of human serum albumin (4% or 20%). Measurements and Main Results: Mice survival, reactivity of mesenteric arteries, and Western blot protein analysis were assessed. Circulating endothelin-1, gluthatione, gluthatione disulfide, and creatinine plasma levels were measured. Nitric oxide production, oxidative, and nitrosative stresses were also measured in situ in endothelial cells. Human serum albumin 4%, but not human serum albumin 20% or normal saline solution, improved survival time of endotoxemic mice. Furthermore, human serum albumin 4% activated endothelial nitric oxide synthase and restored lipopolysaccharide-impaired flow-dependent endothelial dilation in mesenteric arteries. This was associated with a downregulation of nuclear factor κB and an upregulation of nuclear respiratory factor-2 and heme oxygenase-1. Human serum albumin 4% reduced lipopolysaccharide-induced renal dysfunction, enhanced endothelin-1 production and glutathione plasmatic levels, whereas human serum albumin 20% increased gluthatione disulfide. Furthermore, human serum albumin 4% but not 20% blunted lipopolysaccharide-tumor necrosis factor-α-induced oxidative and nitrosative stresses in endothelial cells and increased their gluthatione levels. Conclusions: The present data confirm a protective effect of 4% human serum albumin treatment both on mice survival and endothelial dysfunction by inhibiting inflammatory and oxidative stress pathways induced by endotoxins. Conversely, higher concentrations of human serum albumin were detrimental suggesting a dose-dependent effect

Microparticles Carrying Sonic Hedgehog Favor Neovascularization through the Activation of Nitric Oxide Pathway in Mice

BACKGROUND: Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPs(Shh+)) that are able to regulate in vitro angiogenesis.METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated the ability of MPs(Shh+) to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPs(Shh+), MPs(Shh+) plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPs(Shh+)-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPs(Shh+)-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPs(Shh+) increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPs(Shh+) enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPs(Shh+) on eNOS pathway. Quantitative RT-PCR revealed that MPs(Shh+) treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles. CONCLUSIONS/SIGNIFICANCE: These findings suggest that MPs(Shh+) may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis

Detrimental hemodynamic and inflammatory effects of microparticles originating from septic rats

Objective: Microparticles (MPs) are membrane vesicles with procoagulant and proinflammatory properties released during cell activation and might be potentially involved in the pathophysiology of septic shock. This study was designed to assess the effects of MPs from septic origin on the systemic hemodynamics as well as on the inflammatory, oxidative, and nitrosative stresses.Design: A prospective, randomized, controlled experimental study with repeated measurements. Setting: Investigational animal laboratory. Subjects: Forty healthy rats were randomly allocated to three groups: 10 animals inoculated with MPs isolated from control rats (cMPs), 15 animals inoculated with MPs isolated from sham rats (shMPs), and 15 animals inoculated with MPs isolated from rats with peritonitis (sMPs). Interventions: Rats were anesthetized, mechanically ventilated, and infused with the same amount of cMPs, shMPs, or sMPs. We measured the heart rate, mean arterial pressure, carotid artery, and portal vein blood flows. Hemodynamic parameters were recorded during 7 hours, and then animals were killed. Aorta and heart were harvested for further in vitro tissue analyses. Measurements and Main Results: 1) The cellular origin (phenotype) but not the circulating concentration of MPs was different in septic rats, characterized by a significant increase in leukocyte-derived MPs. 2) sMPs but not cMPs or shMPs decreased mean arterial pressure without any effect on carotid artery and portal vein blood flows. 3) Rats inoculated with sMPs exhibited an increase in superoxide ion production and nuclear factor kappa B activity, overexpression of inducible nitric oxide synthase with subsequent nitric oxide overproduction and decrease in endothelial nitric oxide synthase activation. Conclusions: Rats with sepsis induced by peritonitis exhibited a specific phenotype of MPs. Inoculation of sMPs in healthy rats reproduced hemodynamic, septic inflammatory patterns, associated with oxidative and nitrosative stresses

Three-Dimensional Characterization of the Vascular Bed in Bone Metastasis of the Rat by Microcomputed Tomography (MicroCT)

BackgroundAngiogenesis contributes to proliferation and metastatic dissemination of cancer cells. Anatomy of blood vessels in tumors has been characterized with 2D techniques (histology or angiography). They are not fully representative of the trajectories of vessels throughout the tissues and are not adapted to analyze changes occurring inside the bone marrow cavities. Methodology/Principal Findings We have characterized the vasculature of bone metastases in 3D at different times of evolution of the disease. Metastases were induced in the femur of Wistar rats by a local injection of Walker 256/B cells. Microfil®, (a silicone-based polymer) was injected at euthanasia in the aorta 12, 19 and 26 days after injection of tumor cells. Undecalcified bones (containing the radio opaque vascular casts) were analyzed by microCT, and a first 3D model was reconstructed. Bones were then decalcified and reanalyzed by microCT; a second model (comprising only the vessels) was obtained and overimposed on the former, thus providing a clear visualization of vessel trajectories in the invaded metaphysic allowing quantitative evaluation of the vascular volume and vessel diameter. Histological analysis of the marrow was possible on the decalcified specimens. Walker 256/B cells induced a marked osteolysis with cortical perforations. The metaphysis of invaded bones became progressively hypervascular. New vessels replaced the major central medullar artery coming from the diaphyseal shaft. They sprouted from the periosteum and extended into the metastatic area. The newly formed vessels were irregular in diameter, tortuous with a disorganized architecture. A quantitative analysis of vascular volume indicated that neoangiogenesis increased with the development of the tumor with the appearance of vessels with a larger diameter. Conclusion This new method evidenced the tumor angiogenesis in 3D at different development times of the metastasis growth. Bone and the vascular bed can be identified by a double reconstruction and allowed a quantitative evaluation of angiogenesis upon time

Estrogen Receptor Alpha as a Key Target of Red Wine Polyphenols Action on the Endothelium

BACKGROUND: A greater reduction in cardiovascular risk and vascular protection associated with diet rich in polyphenols are generally accepted; however, the molecular targets for polyphenols effects remain unknown. Meanwhile evidences in the literature have enlightened, not only structural similarities between estrogens and polyphenols known as phytoestrogens, but also in their vascular effects. We hypothesized that alpha isoform of estrogen receptor (ERalpha) could be involved in the transduction of the vascular benefits of polyphenols. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used ERalpha deficient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, Provinols, or delphinidin, an anthocyanin that possesses similar pharmacological profile, is mediated by ERalpha. Indeed, Provinols, delphinidin and ERalpha agonists, 17-beta-estradiol and PPT, are able to induce endothelial vasodilatation in aorta from ERalpha Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing the effects of ERalpha completely prevented the effects of Provinols and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between delphinidin and ERalpha activator site is demonstrated using both binding assay and docking. Most interestingly, the ability of short term oral administration of Provinols to decrease response to serotonin and to enhance sensitivity of the endothelium-dependent relaxation to acetylcholine, associated with concomitant increased NO production and decreased superoxide anions, was completely blunted in ERalpha deficient mice. CONCLUSIONS/SIGNIFICANCE: This study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benefits via ERalpha activation. It is a major breakthrough bringing new insights of the potential therapeutic of polyphenols against cardiovascular pathologies

Biomineralization of amorphous Fe-, Mn- and Si-rich mineral phases by cyanobacteria under oxic and alkaline conditions

Iron and manganese are poorly soluble elements in oxic and alkaline solutions, whereas they are much more soluble under anoxic conditions. As a result, the formation of authigenic mineral phases rich in Fe and/or Mn has traditionally been viewed as diagnostic of global or local anoxic conditions. Here we reveal that some specific cyanobacteria of very small size (&lt; 2 µm, i.e., picocyanobacteria) can biomineralize abundant, authigenic Fe(III)-, Mn(IV)- and Si-rich amorphous phases under oxic conditions in an alkaline lake in Mexico. The resulting biominerals cluster as small globules arranged as rings around the division septum of cyanobacterial cells. These rings are enveloped within an organic, likely polysaccharidic envelope and are partially preserved, at least morphologically, upon sedimentation. Based on their 16S rDNA sequence, these cyanobacteria were affiliated with the Synechococcales order. The high Fe and Mn enrichment of the biominerals questions the systematic inference of anoxic conditions based on their detection. Moreover, this process scavenges iron from the water column, an overlooked biological contribution to the Fe cycle. Finally, it reveals a new case of controlled biomineralization of Si-rich phases by bacteria.</p