Abstract 22: LDLR Promotes and PCSK9 Inhibits LDL-Derived Transintestinal Cholesterol Excretion

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Cedric Le May ◽  
Jean Mathieu Berger ◽  
Bruno Pillot ◽  
Xavier Prieur ◽  
Eric Letessier ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Ldl Receptor ◽  
Cholesteryl Oleate ◽  
Compensatory Mechanism ◽  
Ussing Chambers ◽  
Psc 833 ◽  
Apical Side ◽  
Cholesterol Excretion ◽  
Proximal Intestine

Direct transintestinal cholesterol excretion (TICE) is an alternative path to biliary secretion and accounts for 33% of fecal cholesterol excretion in mice. Objectives: We aimed at identifying i) the lipoproteins involved in TICE ii) the role of intestinal LDL receptor (LDLR) and of its natural inhibitor, circulating proprotein convertase subtilisin kexine type 9 (PCSK9) at the basolateral pole iii) the implication of ATP binding cassette transporter ABCB1ab, a cholesterol floppase localized at the enterocytes apical side. Methods: We labelled human lipoproteins with free 3H cholesterol (free3H) or with 3H-cholesteryl oleate (3HCO). We measured lipoprotein-derived 3H-cholesterol TICE ex vivo in intestinal explants mounted in Ussing chambers. Human explants were obtained from 4 patients undergoing bariatric surgery with their informed consent. In vivo, TICE was measured in mice i.v. injected with radiolabelled lipoproteins, by cannulation of the proximal intestine and concomitant surgical bile diversion, counting radioactivity in intestinal perfusates over 120 minutes. Results: For the first time, we showed direct evidence of TICE in human duodenal explants, from both LDL and HDL. Both lipoproteins (labelled with free3H or 3HCO ) contributed to TICE in mouse explants; TICE was highly responsive to changes in temperature and medium oxygenation. LDL-derived TICE was decreased by 58% (p<0.05) in explants from LDLR knockout mice (LDLR KO), compared with control C57Bl6J. In vivo free3H- or 3HCO-LDL-derived-TICE was conserved in LDLRKO mice, suggestive of a compensatory mechanism. LDL-derived TICE was increased by 62% (p<0.01) in PCSK9KO that present with ∼300% more intestinal LDLR. Acute depletion of intestinal LDLR with purified recombinant PCSK9 (i.v.) led to 40% (p<0.05) less TICE in PCSK9KO but had no effect in LDLRKO, confirming the implication of this receptor. Lovastatin (0.02% W/W 10d) increased TICE by 71% (p < 0.05) in C57Bl6J but not in LDLRKO. Interestingly, using 3H-cholesterol diluted in intralipid as a source, we showed that ABCB1 plays an important role in TICE. Indeed ABCB1ab -/- mice presented with 26% (p<0.05) less TICE than FVB controls in vivo and ABCB1 inhibitor PSC-833 (5mircoM) decreased TICE by 64% in an ABCB1 dependent fashion in explants. Collectively, these results provide the first molecular understanding of TICE.

scholarly journals Regulation of cilia abundance in multiciliated cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rashmi Nanjundappa ◽  
Dong Kong ◽  
Kyuhwan Shim ◽  
Tim Stearns ◽  
Steven L Brody ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Surface Area ◽  
Ex Vivo ◽  
Compensatory Mechanism ◽  
Multiciliated Cells ◽  
Culture Model ◽  
A Cell ◽  
Dependent Mechanism

Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.

scholarly journals Use of Positron Emission Tomography to Study AT1 Receptor Regulation In Vivo

2001 ◽  
Vol 12 (7) ◽  
pp. 1350-1358
Author(s):  
ZSOLT SZABO ◽  
ROBERT C. SPETH ◽  
P. RANDY BROWN ◽  
LEVENTE KERENYI ◽  
PAN FU KAO ◽  
...  
Keyword(s):  
Positron Emission Tomography ◽  
Receptor Binding ◽  
Ex Vivo ◽  
Sodium Intake ◽  
Plasma Renin ◽  
Emission Tomography ◽  
Dietary Sodium ◽  
Compensatory Mechanism ◽  
Positron Emission

Abstract. Increased sodium intake and enhanced sodium sensitivity are implicated in the pathogenesis of hypertension and in the control of a major regulator of BP, the type 1 angiotensin receptor (AT1 receptor). An in vivo technique to study changes of renal AT1 receptors by dietary sodium was developed that uses positron emission tomography (PET). PET revealed that renal cortical AT1 receptor binding was increased in sodium-loaded compared with sodium-deprived dogs, which correlated with ex vivo estimations of AT1 receptor numbers. Plasma renin activity, angiotensin II, and aldosterone were inversely related to changes in AT1 receptor binding. These results demonstrate, for the first time in vivo, that the renal AT1 receptor is inversely related to the activity of the renin angiotensin system, which may provide a compensatory mechanism to prevent inappropriate fluctuations in arterial BP. The ability to measure AT1 receptor binding in vivo has potential significance for clinical studies of AT1 receptors, because PET is a noninvasive imaging technique that is readily applicable in humans.

scholarly journals An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants

2012 ◽  
Vol 302 (4) ◽  
pp. G430-G438 ◽  
Author(s):  
Jenny K. Gustafsson ◽  
Anna Ermund ◽  
Malin E. V. Johansson ◽  
André Schütte ◽  
Gunnar C. Hansson ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Colon ◽  
Epithelial Surface ◽  
Mouse Colon ◽  
Fluorescent Beads ◽  
Apical Side ◽  
Colonic Biopsies ◽  
Small And Large Intestine ◽  
Mouse Ileum

The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ∼40 μm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 μm/h) and mouse (100 μm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 μm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.

scholarly journals A Novel Platelet-Targeting Fibrinolytic Strategy: Endocytosed Urokinase By Megakaryocytes Is Stored in α-Granules and Is Functional In Vivo in a Murine Model of Thrombosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3627-3627
Author(s):  
Hyunsook Ahn ◽  
John Tkaczynski ◽  
Khalil Bdeir ◽  
Victoria Stepanova ◽  
Douglas B. Cines ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Point Of Care ◽  
Ldl Receptor ◽  
Single Amino Acid ◽  
Platelet Glycoprotein ◽  
Single Chain ◽  
Injury Model

We have been interested in developing fibrinolytic agents that have a prolonged half-life and can selectively prevent new thrombi from forming without destabilizing established hemostatic clots. We believe that platelet-targeting of urokinase plasminogen activator (uPA) might be able to achieve this goal. Two approaches had been tried in the past: In the first, we had generated chimeric drugs consisting of a platelet-targeting scFvs fused to a thrombin activatable low molecular weight (LMW) uPA (uPA-T). Infused scFv/uPA-T bound to circulating platelets that targeted the fusion within nascent thrombi where significant amounts of thrombin are generated to activate surface-bound scFv/uPA-T. In contrast, mature thrombi are spared because scFv/uPA-T platelets are not activated on the "shell", nor do they penetrate the "core" where thrombin might be present. Murine studies affirmed αIIbβ3-directed scFv/uPA-T provided thromboprophylaxis, but therapeutic doses caused significant thrombocytopenia in murine and baboon models. We therefore considered a second approach: ectopic storage of uPA during megakaryopoiesis. We found that scuPA was stored in platelet α-granules of transgenic mice that ectopically express single-chain uPA (scuPA) and did not cause systemic fibrinolysis. Infusion of such "scuPA platelets" into wildtype mice was highly effective at preventing new thrombi from developing. We now wish to develop this strategy further by taking advantage of ongoing efforts by others to generate in vitro-grown megakaryocytes (Mks) and platelets that can be modified to express uPA near the point-of-care and then infused into patients, bypassing the need to establish uPA-expressing hematopoietic cell lines. We have previously shown that Mks express low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) during their maturation, whereas the platelets that are released do not. We now asked whether in vitro-grown Mks beginning with CD34+ hematopoietic cells would endocytose uPA as seen in other cell types. We show that Mks internalize and store scuPA (scu-Mks, Fig. 1A), as well as LMW uPA and uPA-T (not shown) after overnight incubation. Endocytosed uPA is found within membrane bound structures that partly colocalize with von Willebrand factor (VWF)-positive granules, suggesting uPA is sorted to α-granules (Fig. 1A). Uptake is blocked by receptor-associated protein (RAP), which inhibits endocytosis by LDL receptor family members, including LRP1. We then studied whether platelets with endocytosed scuPA (scuPA-Plts) prevent nascent thrombus development in immunodeficient NOD-scid IL2rγnull (NSG) mice that are also homozygous for VWFR1326H (a single amino acid substitution that switches species selectivity of VWF so that it binds human platelet glycoprotein (GP) Ib/IX receptor rather than mouse GPIb/IX). These mice show a mild bleeding diathesis in a Rose Bengal photochemical carotid artery injury model unless they are infused with human Mks, which we have shown go on to release functional platelets in the recipient animal in its pulmonary capillary bed over the ensuing several hours (Fig. 1B). Thus, when 106 Mks not exposed to uPA were infused so that ~1-10% of circulating platelets were human, thrombi developed in this model; however similar infusion of scuPA-Mks did not occlude (Fig. 1B). In tail clip studies in the same genotypic mice, where the mice were first corrected with human platelets (Fig. 1C) followed 10 min later by scuPA-Mks, rebleeding did not develop when given a similar dose of scuPA-Mks that prevented thrombosis in the photochemical injury model. These studies suggest that Mks internalize biologically relevant concentrations of uPA through a process likely to involve LRP1. Whether ex vivo loading of Mks with uPA can serve as model for point-of-care therapeutics for thromboprophylaxis and diverse other hematologic and non-hematologic indications should be explored. Disclosures No relevant conflicts of interest to declare.

Alteration of the enterohepatic recirculation of bile acids in rats after exposure to ionizing radiation

2004 ◽  
Vol 82 (2) ◽  
pp. 114-124 ◽  
Author(s):  
P Scanff ◽  
M Souidi ◽  
S Grison ◽  
N M Griffiths ◽  
P Gourmelon
Keyword(s):  
Bile Acid ◽  
Intestinal Absorption ◽  
Bile Acids ◽  
Ex Vivo ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Enterohepatic Recirculation ◽  
Enterohepatic Recycling ◽  
Ussing Chambers

The aim of this work was to study acute alterations of the enterohepatic recirculation (EHR) of bile acids 3 days after an 8-Gy radiation exposure in vivo in the rat by a washout technique. Using this technique in association with HPLC analysis, the EHR of the major individual bile acids was determined in control and irradiated animals. Ex vivo ileal taurocholate absorption was also studied in Ussing chambers. Major hepatic enzyme activities involved in bile acid synthesis were also measured. Measurements of bile acid intestinal content and intestinal absorption efficiency calculation from washout showed reduced intestinal absorption with significant differences from one bile acid to another: absorption of taurocholate and tauromuricholate was decreased, whereas absorption of the more hydrophobic taurochenodeoxycholate was increased, suggesting that intestinal passive diffusion was enhanced, whereas ileal active transport might be reduced. Basal hepatic secretion was increased only for taurocholate, in accordance with the marked increase of CYP8B1 activity in the liver. The results are clearly demonstrate that concomitantly with radiation-induced intestinal bile acid malabsorption, hepatic bile acid synthesis and secretion are also changed. A current working model for pathophysiological changes in enterohepatic recycling after irradiation is thus proposed.Key words: irradiation, bile acids, intestine, liver, enterohepatic recycling.

scholarly journals Bioaccessibility and In Vitro Intestinal Permeability of a Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) Using the Everted Intestine Assay

2021 ◽  
Vol 22 (3) ◽  
pp. 1049
Author(s):  
Lineth Juliana Vega-Rojas ◽  
Ivan Luzardo-Ocampo ◽  
Juan Mosqueda ◽  
Dulce María Palmerín-Carreño ◽  
Antonio Escobedo-Reyes ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Biological Effects ◽  
Intestinal Cell ◽  
Tepary Bean ◽  
Apparent Permeability ◽  
Phaseolus Acutifolius ◽  
Intestinal Membrane ◽  
Apical Side

Tepary bean (Phaseolus acutifolius) lectins exhibit differential in vitro and in vivo biological effects, but their gastrointestinal interactions and digestion have not yet been assessed. This work aimed to evaluate the changes of a recombinant Tepary bean lectin (rTBL-1) through an in vitro and ex vivo gastrointestinal process. A polyclonal antibody was developed to selectively detect rTBL-1 by Western blot (WB) and immunohistochemical analysis. Everted gut sac viability was confirmed until 60 min, where protein bioaccessibility, apparent permeability coefficient, and efflux ratio showed rTBL-1 partial digestion and absorption. Immunoblot assays suggested rTBL-1 internalization, since the lectin was detected in the digestible fraction. The immunohistochemical assay detected rTBL-1 presence at the apical side of the small intestine, potentially due to the interaction with the intestinal cell membrane. The in silico interactions between rTBL-1 and some saccharides or derivatives showed high binding affinity to sialic acid (−6.70 kcal/mol) and N-acetylglucosamine (−6.10 kcal/mol). The ultra-high-performance liquid chromatography–electron spray ionization–quantitative time-of-flight coupled to mass spectrometry (UHPLC–ESI–QTOF/MS) analysis showed rTBL-1 presence in the gastric content and the non-digestible fraction after intestinal simulation conditions. The results indicated that rTBL-1 partially resisted the digestive conditions and interacted with the intestinal membrane, whereas its digestion allowed the absorption or internalization of the protein or the derivative peptides. Further purification of digestion samples should be conducted to identify intact rTBL-1 protein and digested peptides to assess their physiological effects.

scholarly journals Ex vivo and in vitro poultry intestinal models to evaluate antimycotoxins additives

2022 ◽  
Vol 52 (6) ◽  
Author(s):  
Vinicius Duarte ◽  
Adriano Olnei Mallmann ◽  
Camila Tonini ◽  
Diogo Liberalesso ◽  
Cristiane Rosa da Silva ◽  
...  
Keyword(s):  
Intestinal Absorption ◽  
Aflatoxin B1 ◽  
Ex Vivo ◽  
In Vitro Tests ◽  
Intestinal Fluid ◽  
Animal Testing ◽  
Ex Vivo Model ◽  
Ussing Chambers

ABSTRACT: In vitro tests are performed to evaluate the efficacy of antimycotoxins additives (AMAs); nevertheless, such assays show a low correlation with in vivo trials, which are also required to determine AMAs’ efficacy. In search of an alternative method, the current study investigated the use of an ex vivo technique. Six AMAs (AMA1 to AMA6) had their ability to reduce intestinal absorption of aflatoxin B1 (AFB1) evaluated. Jejunal explants were obtained from broilers and subjected to two treatments per AMA in Ussing chambers: T1 (control) - 2.8 mg/L AFB1, and T2 - 2.8 mg/L AFB1 + 0.5% AMA. AMAs were also tested in vitro to assess adsorption of AFB1 in artificial intestinal fluid. In the ex vivo studies, AMA1 to AMA6 decreased intestinal absorption of AFB1 by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively. As for the in vitro results, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. The evaluated ex vivo model proved useful in the assessment of AMAs. No correlation was reported between ex vivo and in vitro findings. Further studies are needed to elucidate the correlation between ex vivo and in vivo results seeking to reduce animal testing.

scholarly journals Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS

Gut ◽  
2019 ◽  
Vol 69 (1) ◽  
pp. 62-73 ◽  
Author(s):  
Shoko Edogawa ◽  
Adam L Edwinson ◽  
Stephanie A Peters ◽  
Lakshmikanth L Chikkamenahalli ◽  
Wendy Sundt ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Healthy Volunteers ◽  
Symptom Severity ◽  
Barrier Function ◽  
Ex Vivo ◽  
Cysteine Protease Inhibitor ◽  
Intestinal Lumen ◽  
Barrier Dysfunction ◽  
Ussing Chambers

ObjectiveThe intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS.Design39 patients with IBS and 25 healthy volunteers completed questionnaires, assessments of in vivo permeability, ex vivo colonic barrier function in Ussing chambers, tight junction (TJ) proteins, ultrastructural morphology and 16 s sequencing of faecal microbiota rRNA. A casein-based assay was used to measure proteolytic activity (PA) in faecal supernatants (FSNs). Colonic barrier function was determined in mice (ex-germ free) humanised with microbial communities associated with different human PA states.ResultsPatients with IBS had higher faecal PA than healthy volunteers. 8/20 postinfection IBS (PI-IBS) and 3/19 constipation- predominant IBS had high PA (>95th percentile). High-PA patients had more and looser bowel movements, greater symptom severity and higher in vivo and ex vivo colonic permeability. High-PA FSNs increased paracellular permeability, decreased occludin and increased phosphorylated myosin light chain (pMLC) expression. Serine but not cysteine protease inhibitor significantly blocked high-PA FSN effects on barrier. The effects on barrier were diminished by pharmacological or siRNA inhibition of protease activated receptor-2 (PAR-2). Patients with high-PA IBS had lower occludin expression, wider TJs on biopsies and reduced microbial diversity than patients with low PA. Mice humanised with high-PA IBS microbiota had greater in vivo permeability than those with low-PA microbiota.ConclusionA subset of patients with IBS, especially in PI-IBS, has substantially high faecal PA, greater symptoms, impaired barrier and reduced microbial diversity. Commensal microbiota affects luminal PA that can influence host barrier function.

scholarly journals Ex Vivo and in Vivo Study of Sucrosomial® Iron Intestinal Absorption and Bioavailability

2018 ◽  
Vol 19 (9) ◽  
pp. 2722 ◽  
Author(s):  
Angela Fabiano ◽  
Elisa Brilli ◽  
Letizia Mattii ◽  
Lara Testai ◽  
Stefania Moscato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Laser Scanning ◽  
Time Course ◽  
Ex Vivo ◽  
Iron Bioavailability ◽  
Laser Scanning Microscopy ◽  
Raw Material ◽  
Confocal Laser ◽  
Ussing Chambers

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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