Lack of WWC2 Protein Leads to Aberrant Angiogenesis in Postnatal Mice

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.

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

Blood ◽

10.1182/blood.v92.11.3997 ◽

1998 ◽

Vol 92 (11) ◽

pp. 3997-4002 ◽

Cited By ~ 92

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.

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

Cellular Physiology and Biochemistry ◽

10.1159/000438645 ◽

2016 ◽

Vol 38 (2) ◽

pp. 487-501 ◽

Cited By ~ 9

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.

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FSH Promotes Rat Follicle Development Through Inhibition of the Hippo Signalling Pathway

10.21203/rs.3.rs-784763/v1 ◽

2021 ◽

Author(s):

Tairen Chen ◽

Mengjing Wu ◽

Yuting Dong ◽

Bin Kong ◽

Yufang Cai ◽

...

Keyword(s):

Western Blot ◽

In Vitro Culture ◽

Granulosa Cells ◽

Signalling Pathway ◽

Control Group ◽

Follicle Development ◽

Follicle Growth ◽

Expression Levels ◽

Hippo Signalling

Abstract Purpose: Whether FSH promotes follicle growth by inhibiting the Hippo signalling pathway.METHODS: Ovaries were cultured in vitro into a control group (no intervention), an FSH group (0.3 IU/mL FSH), and a VP group (10 µg/mL vetiporfin). HE staining and follicle counts were performed at each stage after 3 hours of in vitro culture. Immunohistochemistry was performed to study the expression levels of LATS2, YAP, PLATS2, and PYAP, and their expression levels in each group were also analysed by Western blot.The number of secondary follicles was significantly increased in the FSH group, the arrangement of granulosa cells was neater, the nuclear fixation was reduced, and the number of atretic follicles was decreased in the VP group. The number of secondary follicles was significantly increased, the number of atretic follicles was reduced, and granulosa cell nuclear consolidation was reduced in the VP+FSH group. Immunohistochemistry showed that LATS2 and YAP expression levels were significantly increased and PLATS2 and PYAP expression levels were relatively decreased in the FSH group, PYAP and PLATS2 expression levels were significantly increased and YAP expression was significantly decreased in the VP group, and YAP and LATS2 expression levels were significantly increased and PYAP and PLATS2 expression levels were significantly decreased in the VP+FSH group. By Western blot, LATS2 and YAP were elevated and PYAP and PLAT2 were decreased in the FSH group, LATS2 and YAP were decreased and PYAP and PLATS were significantly elevated in the VP group, and LATS2 and YAP were elevated and PYAP and PLATS2 were decreased in the VP+FSH group.CONCLUSION: FSH promotes follicle development by inhibiting the Hippo signalling pathway.

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

Blood ◽

10.1182/blood.v110.11.3147.3147 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3147-3147 ◽

Cited By ~ 7

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.

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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 ◽

10.1182/blood.v112.11.4098.4098 ◽

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.

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

Blood ◽

10.1182/blood.v92.11.3997.423k52_3997_4002 ◽

1998 ◽

Vol 92 (11) ◽

pp. 3997-4002 ◽

Cited By ~ 5

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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Dose-dependent role of claudin-1 in vivo in orchestrating features of atopic dermatitis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525474113 ◽

2016 ◽

Vol 113 (28) ◽

pp. E4061-E4068 ◽

Cited By ~ 52

Author(s):

Reitaro Tokumasu ◽

Kosuke Yamaga ◽

Yuji Yamazaki ◽

Hiroyuki Murota ◽

Koya Suzuki ◽

...

Keyword(s):

Atopic Dermatitis ◽

Morphological Changes ◽

Contact Hypersensitivity ◽

Expression Level ◽

Barrier Functions ◽

Knock Out ◽

Macrophage Recruitment ◽

Adult Mice ◽

Dose Dependent

Atopic dermatitis (AD) is a chronic inflammatory skin disease in humans. It was recently noted that the characteristics of epidermal barrier functions critically influence the pathological features of AD. Evidence suggests that claudin-1 (CLDN1), a major component of tight junctions (TJs) in the epidermis, plays a key role in human AD, but the mechanism underlying this role is poorly understood. One of the main challenges in studying CLDN1's effects is that Cldn1 knock-out mice cannot survive beyond 1 d after birth, due to lethal dehydration. Here, we established a series of mouse lines that express Cldn1 at various levels and used these mice to study Cldn1’s effects in vivo. Notably, we discovered a dose-dependent effect of Cldn1’s expression in orchestrating features of AD. In our experimental model, epithelial barrier functions and morphological changes in the skin varied exponentially with the decrease in Cldn1 expression level. At low Cldn1 expression levels, mice exhibited morphological features of AD and an innate immune response that included neutrophil and macrophage recruitment to the skin. These phenotypes were especially apparent in the infant stages and lessened as the mice became adults, depending on the expression level of Cldn1. Still, these adult mice with improved phenotypes showed an enhanced hapten-induced contact hypersensitivity response compared with WT mice. Furthermore, we revealed a relationship between macrophage recruitment and CLDN1 levels in human AD patients. Our findings collectively suggest that CLDN1 regulates the pathogenesis, severity, and natural course of human AD.

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IFT-A deficiency in juvenile mice impairs biliary development and exacerbates ADPKD liver disease

10.1101/2020.09.10.289645 ◽

2020 ◽

Author(s):

Wei Wang ◽

Tana S Pottorf ◽

Henry H Wang ◽

Ruochen Dong ◽

Matthew A. Kavanaugh ◽

...

Keyword(s):

Liver Disease ◽

Notch Signaling ◽

Primary Cilia ◽

Intraflagellar Transport ◽

Erk Signaling ◽

Knock Out ◽

Adult Mice ◽

Autosomal Dominant Polycystic Kidney ◽

Knock Out Mice ◽

Cyst Lining

AbstractPolycystic liver disease (PLD) is characterized by the growth of numerous biliary cysts and presents in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD), causing significant morbidity. Interestingly, deletion of intraflagellar transport-B (IFT-B) genes in adult mouse models of ADPKD attenuates severity of PKD and PLD. Here we examine the role of deletion of IFT-A gene, Thm1, in PLD of juvenile and adult Pkd2 conditional knock-out mice. Perinatal deletion of Thm1 results in disorganized and expanded biliary regions, biliary fibrosis, shortened primary cilia on CK19+ biliary epithelial cells, and reduced Notch signaling. In contrast, perinatal deletion of Pkd2 causes PLD, with multiple CK19+ biliary epithelial cell-lined cysts, fibrosis, lengthened primary cilia, and increased Notch and ERK signaling. Perinatal deletion of Thm1 in Pkd2 conditional knock-out mice increased hepatomegaly and liver necrosis, indicating enhanced liver disease severity. In contrast to effects in the developing liver, deletion of Thm1 in adult mice, alone and together with Pkd2, did not cause a biliary phenotype nor affect Pkd2-mutant PLD, respectively. However, similar to juvenile PLD, Notch and ERK signaling were increased in adult Pkd2-mutant cyst-lining cholangiocytes. Taken together, Thm1 is required for biliary tract development, likely by enabling Notch signaling, and proper biliary development restricts PLD severity. Unlike IFT-B genes, Thm1 does not affect hepatic cystogenesis, suggesting divergent regulation of signaling and cystogenic processes in the liver by IFT-B and –A. Notably, increased Notch signaling in cyst-lining cholangiocytes may indicate that aberrant activation of this pathway promotes hepatic cystogenesis, presenting as a novel potential therapeutic target.

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Identification of a developmental switch in information transfer between whisker S1 and S2 cortex in mice

10.1101/2021.08.06.455364 ◽

2021 ◽

Author(s):

Theofanis Karayannis ◽

Linbi Cai ◽

Jenq-Wei Yang ◽

Shen-Ju Chou ◽

Chia-Fang Wang ◽

...

Keyword(s):

Information Transfer ◽

Barrel Cortex ◽

Wide Field ◽

Sensory Stimuli ◽

Knock Out ◽

Electrophysiological Recordings ◽

Primary Sensory Cortex ◽

Animal Navigation ◽

Knock Out Mice

The whiskers of rodents are a key sensory organ that provides critical tactile information for animal navigation and object exploration throughout life. Previous work has explored the developmental sensory-driven activation of the primary sensory cortex processing whisker information (wS1), also called barrel cortex. This body of work has shown that the barrel cortex is already activated by sensory stimuli during the first post-natal week. However, it is currently unknown when over the course of development these stimuli begin being processed by higher order cortical areas, such as secondary whisker somatosensory area (wS2). Here we investigate for the first time the developmental engagement of wS2 by sensory stimuli and the emergence of cortico-cortical communication from wS1 to wS2. Using in vivo wide-field imaging and electrophysiological recordings in control and conditional knock-out mice we find that wS1 and wS2 are able to process bottom-up information coming from the thalamus already right after birth. We identify that it is only at the end of the first post-natal week that wS1 begins to provide excitation into wS2, a connection which begins to acquire feed-forward inhibition characteristics after the second post-natal week. Therefore, we have uncovered a developmental window during which excitatory versus inhibitory functional connectivity between wS1 and wS2 takes place.

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Cholesterol Regulates Exosome Release in Cultured Astrocytes

Frontiers in Immunology ◽

10.3389/fimmu.2021.722581 ◽

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.

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