scholarly journals Cholesterol Regulates Exosome Release in Cultured Astrocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohammad Abdullah ◽  
Tomohisa Nakamura ◽  
Taslima Ferdous ◽  
Yuan Gao ◽  
Yuxin Chen ◽  
...  
Keyword(s):  
High Fat Diet ◽  
Normal Diet ◽  
Cellular Cholesterol ◽  
Knock Out ◽  
Pi3k Inhibitor Ly294002 ◽  
Akt Phosphorylation ◽  
Cholesterol Levels ◽  
Cultured Astrocytes ◽  
Knock Out Mice

Exosomes are vesicles secreted by various kinds of cells, and they are rich in cholesterol, sphingomyelin (SM), phosphatidylcholine, and phosphatidylserine. Although cellular sphingolipid-mediated exosome release has been reported, the involvement of other lipid components of cell membranes in the regulation of exosome release is poorly understood. Here, we show that the level of exosome release into conditioned media is significantly reduced in cultured astrocytes prepared from apolipoprotein E (ApoE) knock-out mice when compared to those prepared from wild-type (WT) mice. The reduced level of exosome release was accompanied by elevated levels of cellular cholesterol. The addition of cholesterol to WT astrocytes significantly increased the cellular cholesterol levels and reduced exosome release. PI3K/Akt phosphorylation was enhanced in ApoE-deficient and cholesterol-treated WT astrocytes. In contrast, the depletion of cholesterol in ApoE-deficient astrocytes due to treatment with β-cyclodextrin recovered the exosome release level to a level similar to that in WT astrocytes. In addition, the reduced levels of exosome release due to the addition of cholesterol recovered to the control levels after treatment with a PI3K inhibitor (LY294002). The cholesterol-dependent regulation of exosome release was also confirmed by in vivo experiments; that is, exosome levels were significantly reduced in the CSF and blood serum of WT mice that were fed a high-fat diet and had increased cholesterol levels when compared to those in WT mice that were fed a normal diet. These results suggest that exosome release is regulated by cellular cholesterol via stimulation of the PI3K/Akt signal pathway.

Abstract 114: Cytokine Response to Diet and Exercise Affects Atheromatous Matrix Metalloproteinase-2/9 Activity in Mice

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Dong-Eog Kim
Keyword(s):  
Matrix Metalloproteinase ◽  
Visceral Fat ◽  
Near Infrared ◽  
Normal Diet ◽  
Matrix Metalloproteinase 2 ◽  
Knock Out ◽  
Diet And Exercise ◽  
Knock Out Mice ◽  
Inflammatory Milieu

Objective: To identify the principal circulating factors that modulate atheromatous matrix metalloproteinase (MMP) activity in response to diet and exercise. Methods and Results: Apolipoprotein-E knock-out mice (n=56) with preexisting plaque, fed either Western diet (WD) or normal diet (ND), underwent either 10-week treadmill exercise or not. In vivo atheromatous MMP activity was visualized using molecular imaging with an MMP-2/9 activatable near-infrared-fluorescent probe. We measured atherosclerosis-related cytokines, lipid levels, visceral fat, and correlated these outcome measures to atheromatous MMP activity. Body weight, visceral fat, and plaque size were all higher in WD-fed animals than in ND-fed animals. Exercise training did not significantly affect these parameters in either WD-fed animals or ND-fed animals. However, atheromatous MMP activity was different: ND animals with and without exercise had similar low MMP activities, WD animals without exercise had high MMP activity, and WD animals with exercise had reduced levels of MMP activity, close to the levels of ND animals. Factor analysis and path analysis showed that soluble vascular cell adhesion molecule (sVCAM)-1 was directly positively related to atheromatous MMP activity. Adiponectin was indirectly negatively related to atheromatous MMP activity by way of sVCAM-1. Resistin was indirectly positively related to atheromatous MMP activity by way of sVCAM-1. In addition, visceral fat amount was indirectly positively associated with atheromatous MMP activity, by way of adiponectin reduction and resistin elevation. Conclusion: Diet and exercise affects atheromatous MMP activity by modulating the systemic inflammatory milieu, with sVCAM-1, resistin, and adiponectin closely interacting with each other and with visceral fat.

scholarly journals Metabolic consequences for mice lacking Endosialin: LC–MS/MS-based metabolic phenotyping of serum from C56Bl/6J Control and CD248 knock‐out mice

Metabolomics ◽  
2021 ◽  
Vol 17 (2) ◽  
Author(s):  
Emily G. Armitage ◽  
Alan Barnes ◽  
Kieran Patrick ◽  
Janak Bechar ◽  
Matthew J. Harrison ◽  
...  
Keyword(s):  
High Fat Diet ◽  
High Performance ◽  
Normal Diet ◽  
High Fat ◽  
Metabolic Phenotyping ◽  
Knock Out ◽  
Flight Mass Spectrometry ◽  
Lipid Species ◽  
Metabolic Consequence ◽  
Knock Out Mice

Abstract Introduction The Endosialin/CD248/TEM1 protein is expressed in adipose tissue and its expression increases with obesity. Recently, genetic deletion of CD248 has been shown to protect mice against atherosclerosis on a high fat diet. Objectives We investigated the effect of high fat diet feeding on visceral fat pads and circulating lipid profiles in CD248 knockout mice compared to controls. Methods From 10 weeks old, CD248−/− and +/+ mice were fed either chow (normal) diet or a high fat diet for 13 weeks. After 13 weeks the metabolic profiles and relative quantities of circulating lipid species were assessed using ultra high performance liquid chromatography-quadrupole time-of flight mass spectrometry (UHPLC–MS) with high resolution accurate mass (HRAM) capability. Results We demonstrate a specific reduction in the size of the perirenal fat pad in CD248−/− mice compared to CD248+/+, despite similar food intake. More strikingly, we identify significant, diet-dependent differences in the serum metabolic phenotypes of CD248 null compared to age and sex-matched wildtype control mice. Generalised protection from HFD-induced lipid accumulation was observed in CD248 null mice compared to wildtype, with particular reduction noted in the lysophosphatidylcholines, phosphatidylcholines, cholesterol and carnitine. Conclusions Overall these results show a clear and protective metabolic consequence of CD248 deletion in mice, implicating CD248 in lipid metabolism or trafficking and opening new avenues for further investigation using anti-CD248 targeting agents.

scholarly journals Lack of WWC2 Protein Leads to Aberrant Angiogenesis in Postnatal Mice

2021 ◽  
Vol 22 (10) ◽  
pp. 5321
Author(s):  
Viktoria Constanze Brücher ◽  
Charlotte Egbring ◽  
Tanja Plagemann ◽  
Pavel I. Nedvetsky ◽  
Verena Höffken ◽  
...  
Keyword(s):  
Signalling Pathway ◽  
Control Group ◽  
Knock Out ◽  
Ocular Angiogenesis ◽  
Sprouting Angiogenesis ◽  
Adult Mice ◽  
Upstream Regulator ◽  
Knock Out Mice ◽  
Hippo Signalling

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 3997-4002 ◽  
Author(s):  
Dirk Meyer ◽  
Carsten Schiller ◽  
Jürgen Westermann ◽  
Shozo Izui ◽  
Wouter L. W. Hazenbos ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Autoimmune Hemolytic Anemia ◽  
Knock Out ◽  
Effector Cells ◽  
Igg Isotypes ◽  
Knock Out Mice ◽  
Deficient Mice ◽  
Experimental Autoimmune

Abstract In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

scholarly journals Pannexin-1 Deficient Mice Have an Increased Susceptibility for Atrial Fibrillation and Show a QT-Prolongation Phenotype

2016 ◽  
Vol 38 (2) ◽  
pp. 487-501 ◽  
Author(s):  
Stella Petric ◽  
Sofia Klein ◽  
Lisa Dannenberg ◽  
Tillman Lahres ◽  
Lukas Clasen ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Cardiac Electrophysiology ◽  
Electrophysiological Study ◽  
Atp Release ◽  
Release Channel ◽  
Knock Out ◽  
Pannexin 1 ◽  
Significant Prolongation ◽  
Knock Out Mice

Background/Aims: Pannexin-1 (Panx1) is an ATP release channel that is ubiquitously expressed and coupled to several ligand-gated receptors. In isolated cardiac myocytes, Panx1 forms large conductance channels that can be activated by Ca2+ release from the sarcoplasmic reticulum. Here we characterized the electrophysiological function of these channels in the heart in vivo, taking recourse to mice with Panx1 ablation. Methods: Cardiac phenotyping of Panx1 knock-out mice (Panx1-/-) was performed by employing a molecular, cellular and functional approach, including echocardiography, surface and telemetric ECG recordings with QT analysis, physical stress testing and quantification of heart rate variability. In addition, an in vivo electrophysiological study entailed programmed electrical stimulation using an intracardiac octapolar catheter. Results: Panx1 deficiency results in a higher incidence of AV-block, delayed ventricular depolarisation, significant prolongation of QT- and rate corrected QT-interval and a higher incidence of atrial fibrillation after intraatrial burst stimulation. Conclusion: Panx1 seems to play an important role in murine cardiac electrophysiology and warrants further consideration in the context of hereditary forms of atrial fibrillation.

In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Peter L. Turecek ◽  
Jürgen Siekmann ◽  
Herbert Gritsch ◽  
Katalin Váradi ◽  
Rafi-Uddin Ahmad ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Full Length ◽  
Thrombin Inhibitor ◽  
Knock Out ◽  
Knock Out Mice

Abstract Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.

Recombinant Human Coagulation Factor IX Expressed in HEK293 Cells: Influence of Serin Phosphorylation and Tyrosine Sulfation on Pharmacokinetic Properties in FIX-Knock-out Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4098-4098
Author(s):  
Ernst Boehm ◽  
Michael Dockal ◽  
Meinhard Hasslacher ◽  
Artur Mitterer ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hek293 Cells ◽  
High Yield ◽  
High Similarity ◽  
Tyrosine Sulfation ◽  
Knock Out ◽  
Pharmacokinetic Properties ◽  
Knock Out Mice ◽  
In Vivo Recovery

Abstract Recombinant factor IX (rFIX) expressed in Chinese hamster ovary (CHO) cells has been shown to be safe and effective in clinical studies, but differs in pharmacokinetics from plasma-derived FIX (pdFIX). In clinical studies, CHO-derived rFIX had a 30–50 % lower in-vivo recovery when compared to pdFIX, whereas mean residence time and terminal half-life did not differ between preparations. Although rFIX shows high similarity to pdFIX in structure and function, differences in glycosylation and gamma-carboxylation degree can be detected. Moreover, although experimental proof has yet to be published, the lower degree of phosphorylation of amino acid serine 155, and the lower degree of sulfation of tyrosine 158 have been hypothesized to be causative for the lower in-vivo recovery of rFIX. These two modifications occur at less than 20 % for the tyrosine-sulfation and at less than 1 % for the serine phosphorylation in rFIX, whereas pdFIX has both modifications to more than 90 % completed. We identified human HEK293 cells to perform rFIX phosphorylation and sulfation to a higher extent than CHO cells. A rFIX-producing cell line derived from HEK293 cells was generated by stable transfection, and was adapted to suspension culture conditions to allow lab-scale fermentation. rFIX was produced and purified from a single fermentation run using two different down-stream process schemes: the first was able to enrich high-phosphorylated and -sulfated rFIX; the second to purify total rFIX from the supernatant at high yield. For pharmacokinetic comparison, these HEK293 materials, CHO-derived rFIX, and a pdFIX preparation were formulated in the same buffer. Determination of phosphorylation and sulfation by mass spectrometry showed a phosphorylation and sulfation degree of 50 % plus a 20 % single modification (phosphorylation or sulfation) for the HEK293-material purified by the modification enrichment method versus 15 % for both modifications plus a 15 % single modification for the material purified by the high-yield protocol. The values for CHO-derived rFIX and pdFIX were similar to those in the literature. Oligosaccharide mapping revealed glycosylation differences among CHO-, HEK293-, and pdFIX preparations, but high similarity between both HEK293-derived materials. We compared the pharmacokinetics of the various FIX preparations in FIX-knock-out mice. In-vivo recovery and area under the curve were statistically significantly higher for the high phosphorylated and sulfated HEK293-material than for total rFIX derived from HEK293 cells. However, these two parameters were lower for both HEK293-derived rFIX preparations than for CHO-derived rFIX, and lower for CHO-derived rFIX than for pdFIX. This may be due to glycosylation differences between these FIX preparations. Mean residence times and terminal half-lives were similar for all preparations. In summary, these findings emphasize that the degree of rFIX-sulfation and -phosphorylation influences the pharmacokinetic properties of rFIX.

FcγRIII (CD16)-Deficient Mice Show IgG Isotype-Dependent Protection to Experimental Autoimmune Hemolytic Anemia

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 3997-4002 ◽  
Author(s):  
Dirk Meyer ◽  
Carsten Schiller ◽  
Jürgen Westermann ◽  
Shozo Izui ◽  
Wouter L. W. Hazenbos ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Autoimmune Hemolytic Anemia ◽  
Knock Out ◽  
Effector Cells ◽  
Igg Isotypes ◽  
Knock Out Mice ◽  
Deficient Mice ◽  
Experimental Autoimmune

In autoimmune hemolytic anemia (AIHA), there is accumulating evidence for an involvement of FcγR expressed by phagocytic effector cells, but demonstration of a causal relationship between individual FcγRs and IgG isotypes for disease development is lacking. Although the relevance of IgG isotypes to human AIHA is limited, we could show a clear IgG isotype dependency in murine AIHA using pathogenic IgG1 (105-2H) and IgG2a (34-3C) autoreactive anti–red blood cell antibodies in mice defective for FcγRIII, and comparing the clinical outcome to those in wild-type mice. FcγRIII-deficient mice were completely resistent to the pathogenic effects of 105-2H monoclonal antibody, as shown by a lack of IgG1-mediated erythrophagocytosis in vitro and in vivo. In addition, the IgG2a response by 34-3C induced a less severe but persistent AIHA in FcγRIII knock-out mice, as documented by a decrease in hematocrit. Blocking studies indicated that the residual anemic phenotype induced by 34-3C in the absence of FcγRIII reflects an activation of FcγRI that is normally coexpressed with FcγRIII on macrophages. Together these results show that the pathogenesis of AIHA through IgG1-dependent erythrophagocytosis is exclusively mediated by FcγRIII and further suggest that FcγRI, in addition to FcγRIII, contributes to this autoimmune disease when other IgG isotypes such as IgG2a are involved.

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