Abnormal Lysosomal Positioning and Small Extracellular Vesicle Secretion in Arterial Stiffening and Calcification of Mice Lacking Mucolipin 1 Gene

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1−/− mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1−/− mice as compared to their wild-type littermates. Besides, Mcoln1−/− mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1−/− mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1−/− mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

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Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice

Scientific Reports ◽

10.1038/s41598-020-58568-5 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Owais M. Bhat ◽

Guangbi Li ◽

Xinxu Yuan ◽

Dandan Huang ◽

Erich Gulbins ◽

...

Keyword(s):

Smooth Muscle ◽

Knockout Mice ◽

Gene Knockout ◽

Extracellular Vesicle ◽

Vesicle Release ◽

Gene Knockout Mice ◽

Arterial Medial Calcification ◽

Medial Calcification

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Prominent Indomethacin-Induced Enteropathy in Fcgriib Defi-cient lupus Mice: An Impact of Macrophage Responses and Immune Deposition in Gut

International Journal of Molecular Sciences ◽

10.3390/ijms22031377 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1377

Author(s):

Thansita Bhunyakarnjanarat ◽

Kanyarat Udompornpitak ◽

Wilasinee Saisorn ◽

Bhumdhanin Chantraprapawat ◽

Peerapat Visitchanakun ◽

...

Keyword(s):

Cytokine Production ◽

High Dose ◽

Flux Analysis ◽

Wild Type ◽

Fc Gamma Receptor ◽

Serum Cytokines ◽

Extracellular Flux ◽

Gamma Receptor ◽

Gut Leakage ◽

Gastrointestinal Permeability

A high dose of NSAIDs, a common analgesic, might induce lupus activity through several NSAIDs adverse effects including gastrointestinal permeability defect (gut leakage) and endotoxemia. Indomethacin (25 mg/day) was orally administered for 7 days in 24-wk-old Fc gamma receptor IIb deficient (FcgRIIb-/-) mice, an asymptomatic lupus model (increased anti-dsDNA without lupus nephritis), and age-matched wild-type (WT) mice. Severity of indomethacin-induced enteropathy in FcgRIIb-/- mice was higher than WT mice as demonstrated by survival analysis, intestinal injury (histology, immune-deposition, and intestinal cytokines), gut leakage (FITC-dextran assay and endotoxemia), serum cytokines, and lupus characteristics (anti-dsDNA, renal injury, and proteinuria). Prominent responses of FcgRIIb-/- macrophages toward lipopolysaccharide (LPS) compared to WT cells due to the expression of only activating-FcgRs without inhibitory-FcgRIIb were demonstrated. Extracellular flux analysis indicated the greater mitochondria activity (increased respiratory capacity and respiratory reserve) in FcgRIIb-/- macrophages with a concordant decrease in glycolysis activity when compared to WT cells. In conclusion, gut leakage-induced endotoxemia is more severe in indomethacin-administered FcgRIIb-/- mice than WT, possibly due to the enhanced indomethacin toxicity from lupus-induced intestinal immune-deposition. Due to a lack of inhibitory-FcgRIIb expression, mitochondrial function, and cytokine production of FcgRIIb-/- macrophages were more prominent than WT cells. Hence, lupus disease-activation from NSAIDs-enteropathy-induced gut leakage is possible.

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Mutation of the 5' untranslated region stem-loop mRNA structure reduces type I collagen deposition and arterial stiffness in male obese mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00076.2021 ◽

2021 ◽

Author(s):

Francisco I. Ramirez-Perez ◽

Makenzie L. Woodford ◽

Mariana Morales-Quinones ◽

Zachary I. Grunewald ◽

Francisco J Cabral-Amador ◽

...

Keyword(s):

Insulin Resistance ◽

Type 2 Diabetes ◽

Arterial Stiffness ◽

Type I Collagen ◽

Type I ◽

Collagen Deposition ◽

Wild Type ◽

Stem Loop ◽

Arterial Stiffening

Arterial stiffening, a characteristic feature of obesity and type 2 diabetes, contributes to the development and progression of cardiovascular diseases (CVD). Currently, no effective prophylaxis or therapeutics is available to prevent or treat arterial stiffening. A better understanding of the molecular mechanisms underlying arterial stiffening is vital to identify newer targets and strategies to reduce CVD burden. A major contributor to arterial stiffening is increased collagen deposition. In the 5' untranslated regions of mRNAs encoding for type I collagen, an evolutionally conserved stem-loop (SL) structure plays an essential role in its stability and post-transcriptional regulation. Here, we show that feeding a high fat/high sucrose (HFHS) diet for 28 weeks increases adiposity, insulin resistance, and blood pressure in male wild-type littermates. Moreover, arterial stiffness, assessed in vivo via aortic pulse wave velocity, and ex vivo using atomic force microscopy in aortic explants or pressure myography in isolated femoral and mesenteric arteries, was also increased in those mice. Notably, all these indices of arterial stiffness, along with collagen type I levels in the vasculature, were reduced in HFHS-fed mice harboring a mutation in the 5'SL structure, relative to wild-type littermates. This protective vascular phenotype in 5'SL-mutant mice did not associate with a reduction in insulin resistance or blood pressure. These findings implicate the 5'SL structure as a putative therapeutic target to prevent or reverse arterial stiffening and CVD associated with obesity and type 2 diabetes.

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High-Throughput Single-Cell Extracellular Vesicle Secretion Analysis on a Desktop Scanner without Cell Counting

Analytical Chemistry ◽

10.1021/acs.analchem.1c01446 ◽

2021 ◽

Author(s):

Fengjiao Zhu ◽

Yahui Ji ◽

Linmei Li ◽

Xue Bai ◽

Xianming Liu ◽

...

Keyword(s):

Single Cell ◽

High Throughput ◽

Extracellular Vesicle ◽

Cell Counting ◽

Vesicle Secretion

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Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

Journal of General Virology ◽

10.1099/vir.0.035378-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2838-2848 ◽

Cited By ~ 56

Author(s):

Shigeo Nagashima ◽

Masaharu Takahashi ◽

Suljid Jirintai ◽

Toshinori Tanaka ◽

Tsutomu Nishizawa ◽

...

Keyword(s):

Susceptibility Gene ◽

Multivesicular Body ◽

Hepatitis E ◽

Negative Control ◽

Dominant Negative ◽

Wild Type ◽

Orf3 Protein ◽

Virus Particles ◽

Virion Release ◽

Vacuolar Protein

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles.

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The forces driving cancer extracellular vesicle secretion

Neoplasia ◽

10.1016/j.neo.2020.11.011 ◽

2021 ◽

Vol 23 (1) ◽

pp. 149-157

Author(s):

Maarten P. Bebelman ◽

Eline Janssen ◽

D. Michiel Pegtel ◽

Caitrin Crudden

Keyword(s):

Extracellular Vesicle ◽

Vesicle Secretion

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Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1997.272.2.r563 ◽

1997 ◽

Vol 272 (2) ◽

pp. R563-R569 ◽

Cited By ~ 19

Author(s):

L. R. Leon ◽

W. Kozak ◽

J. Peschon ◽

M. J. Kluger

Keyword(s):

Body Weight ◽

Food Intake ◽

Motor Activity ◽

Knockout Mice ◽

Low Dose ◽

Late Phase ◽

High Dose ◽

Wild Type ◽

Inflammatory Stimuli ◽

Necrosis Factor

We examined the effects of injections of systemic [lipopolysaccharide (LPS), 2.5 mg/kg or 50 pg/kg ip] or local (turpentine, 100 microl sc) inflammatory stimuli on fever, motor activity, body weight, and food intake in tumor necrosis factor (TNF) double receptor (TNFR)-knockout mice. A high dose of LPS resulted in exacerbated fevers in TNFR-knockout mice compared with wild-type mice for the early phase of fever (3-15 h); the late phase of fever (16-24 h) and fevers to a low dose of LPS were similar in both groups. Motor activity, body weight, and food intake were similarly reduced in both groups of mice after LPS administration. In response to turpentine, TNFR-knockout and wild-type mice developed virtually identical responses to all variables monitored. These results suggest that 1) TNF modulates fevers to LPS dose dependently, 2) TNF does not modulate fevers to a subcutaneous injection of turpentine, and 3) knockout mice may develop cytokine redundancy in the regulation of the acute phase response to intraperitoneally injected LPS or subcutaneously injected turpentine.

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