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2022 ◽  
Vol 146 ◽  
pp. 112486
Author(s):  
Shiyu Li ◽  
Yusong Jin ◽  
Wenyi Fu ◽  
Abigail D. Cox ◽  
Dale Lee ◽  
...  

Author(s):  
Fang-Hao Guo ◽  
Ya-Na Guan ◽  
Jun-Jun Guo ◽  
Lu-Jun Zhang ◽  
Jing-Jing Qiu ◽  
...  

Background: Endothelial cells (ECs) play a critical role in angiogenesis and vascular remodeling. The heterogeneity of ECs has been reported at adult stages, yet it has not been fully investigated. This study aims to assess the transcriptional heterogeneity of developmental ECs at spatiotemporal level and to reveal the changes of embryonic ECs clustering when endothelium-enriched microRNA-126 (miR-126) was specifically knocked out. Methods: C57BL/6J mice embryos at day 14.5 were harvested and digested, followed by fluorescence-activated cell sorting to enrich ECs. Then, single-cell RNA sequencing was applied to enriched embryonic ECs. Tie2 (Tek receptor tyrosine kinase)-cre–mediated ECs-specific miR-126 knockout mice were constructed, and ECs from Tie2-cre–mediated ECs-specific miR-126 knockout embryos were subjected to single-cell RNA sequencing. Results: Embryonic ECs were clustered into 11 groups corresponding to anatomic characteristics. The vascular bed (arteries, capillaries, veins, lymphatics) exhibited transcriptomic similarity across the developmental stage. Embryonic ECs had higher proliferative potential than adult ECs. Integrating analysis showed that 3 ECs populations (hepatic, mesenchymal transition, and pulmonary ECs) were apparently disorganized after miR-126 being knocked out. Gene ontology analysis revealed that disrupted ECs were mainly related to hypoxia, glycometabolism, and vascular calcification. Additionally, in vivo experiment showed that Tie2-cre–mediated ECs-specific miR-126 knockout mice exhibited excessive intussusceptive angiogenesis; reductive glucose and pyruvate tolerance; and excessive accumulation of calcium. Agonist miR-126-3p agomir significantly rescued the phenotype of glucose metabolic dysfunction in Tie2-cre–mediated ECs-specific miR-126 knockout mice. Conclusions: The heterogeneity of ECs is established as early as the embryonic stage. The deficiency of miR-126 disrupts the differentiation and diversification of embryonic ECs, suggesting that miR-126 plays an essential role in the maintenance of ECs heterogeneity.


Author(s):  
Xiaodong Jiao ◽  
Zhiwei Ma ◽  
Jingqi Lei ◽  
Pinghu Liu ◽  
Xiaoyu Cai ◽  
...  

Purpose: We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K+ channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials.Methods: A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter. Lentiviral replacement of Kcnj13 was driven by the EF1a or VMD2 promoters.Results: Blue-Gal expression is evident in E12.5 brain ventricular choroid plexus, lens, neural retina layer, and anterior RPE. In the adult eye expression is seen in the ciliary body, RPE and choroid. Adult conditional Kcnj13 ko mice show loss of photoreceptors in the outer nuclear layer, inner nuclear layer thinning with loss of bipolar cells, and thinning and disruption of the outer plexiform layer, correlating with Cre expression in the overlying RPE which, although preserved, shows morphological disruption. Fundoscopy and OCT show signs of retinal degeneration consistent with the histology, and photopic and scotopic ERGs are decreased in amplitude or extinguished. Lentiviral based replacement of Kcnj13 resulted in increased ERG c- but not a- or b- wave amplitudes.Conclusion: Ocular KCNJ13 expression starts in the choroid, lens, ciliary body, and anterior retina, while later expression centers on the RPE with no/lower expression in the neuroretina. Although KCNJ13 expression is not required for survival of the RPE, it is necessary for RPE maintenance of the photoreceptors, and loss of the photoreceptor, outer plexiform, and outer nuclear layers occur in adult KCNJ13 cKO mice, concomitant with decreased amplitude and eventual extinguishing of the ERG and signs of retinitis pigmentosa on fundoscopy and OCT. Kcnj13 replacement resulting in recovery of the ERG c- but not a- and b-waves is consistent with the degree of photoreceptor degeneration seen on histology.


2022 ◽  
Author(s):  
Jane A Mitchell ◽  
Maria Vinokurova ◽  
Maria E Lopes-Pires ◽  
Fisnik Shala ◽  
Paul C Armstrong ◽  
...  

Prostacyclin is an anti-thrombotic hormone long considered to be derived from the vascular endothelium. However, the role of non-vascular sources for prostacyclin synthesis has not been systematically evaluated due to a lack of tools. Here we used cell-specific knockout mice and human tissues to show that lung, and other tissues, are powerful producers of prostacyclin independent of their vascular components. Instead, in mice and humans, lung prostacyclin synthesis is associated with fibroblasts. The fibroblast-derived prostaglandins enter the circulation and provide systemic anti-thrombotic protection. These observations define a new paradigm in prostacyclin biology in which fibroblast/non-vascular-derived prostacyclin works in parallel with prostaglandins produced by the endothelium to control cardiovascular health. These results may explain how local diseases of the lung and elsewhere result in cardiovascular risk.


2022 ◽  
Author(s):  
Raquel Guillamat-Prats ◽  
Daniel Hering ◽  
Martina Rami ◽  
Carmen Haerdner ◽  
Donato Santovito ◽  
...  

Abstract Identifying novel pathways regulating the adaptive immune response in chronic inflammatory diseases such as atherosclerosis is of particular interest in view of developing new therapeutic drugs. Here we report that the lipid receptor GPR55 is highly expressed by splenic B cells and inversely correlates with atheroma plaque size in mice. In human carotid endarterectomy specimen, GPR55 transcript levels were significantly lower in unstable compared to stable carotid plaques. To study the impact of GPR55 deficiency in atherosclerosis, we crossed Gpr55 knockout mice with apolipoprotein E (ApoE) knockout mice and subjected the mice to Western diet for 4 to 16 weeks. Compared to ApoE-/- controls, ApoE-/-Gpr55-/- mice developed larger plaques with increased necrotic core size, associated with elevated circulating and aortic leukocyte counts. Flow cytometry, immunofluorescence and RNA-sequencing analysis of splenic B cells in these mice revealed a hyperactivated B cell phenotype with disturbed plasma cell maturation and immunoglobulin (Ig)G antibody overproduction. The specific contribution of B cell GPR55 in atherosclerosis was further studied in mixed Gpr55-/-/µMT bone marrow chimeras on low density receptor deficiency (Ldlr-/-) background, revealing that B-cell specific depletion of Gpr55 was sufficient to promote plaque development. Conversely, adoptive transfer of wildtype B cells into ApoE-/-Gpr55-/- mice blunted the proatherogenic phenotype. In vitro stimulation of splenocytes with the endogenous GPR55 ligand LPI promoted plasma cell proliferation and enhanced B cell activation marker expression, which was inhibited by the GPR55 antagonist CID16020046. Collectively, these discoveries provide new evidence for GPR55 as key modulator of the adaptive immune response in atherosclerosis. Targeting GPR55 could be useful to limit inflammation and plaque progression in patients suffering from atherosclerosis.


Biology ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 123
Author(s):  
Abdel A. Alli ◽  
Ling Yu ◽  
Ewa Wlazlo ◽  
Sadat Kasem ◽  
Mohammed F. Gholam ◽  
...  

Hypertension is associated with an increased renal expression and activity of the epithelial sodium channel (ENaC) and iron deficiency. Distal tubules absorb iron, causing perturbations that may influence local responses. In this observational study, we investigated the relationship between iron content and ENaC expression and activity using two cell lines and hepcidin knockout mice (a murine model of iron overload). We found that iron did not transcriptionally regulate ENaC in hepcidin knockout mice or in vitro in collecting duct cells. However, the renal tubules of hepcidin knockout mice have a lower expression of ENaC protein. ENaC activity in cultured Xenopus 2F3 cells and mpkCCD cells was inhibited by iron, which could be reversed by iron chelation. Thus, our novel findings implicate iron as a regulator of ENaC protein and its activity.


Author(s):  
Takafumi Matsumura ◽  
Taichi Noda ◽  
Yuhkoh Satouh ◽  
Akane Morohoshi ◽  
Shunsuke Yuri ◽  
...  

Fertilization occurs as the culmination of multi-step complex processes. First, mammalian spermatozoa undergo the acrosome reaction to become fusion-competent. Then, the acrosome-reacted spermatozoa penetrate the zona pellucida and adhere to and finally fuse with the egg plasma membrane. IZUMO1 is the first sperm protein proven to be essential for sperm-egg fusion in mammals, as Izumo1 knockout mouse spermatozoa adhere to but fail to fuse with the oolemma. However, the IZUMO1 function in other species remains largely unknown. Here, we generated Izumo1 knockout rats by CRISPR/Cas9 and found the male rats were infertile. Unlike in mice, Izumo1 knockout rat spermatozoa failed to bind to the oolemma. Further investigation revealed that the acrosome-intact sperm binding conceals a decreased number of the acrosome-reacted sperm bound to the oolemma in Izumo1 knockout mice. Of note, we could not see any apparent defects in the binding of the acrosome-reacted sperm to the oolemma in the mice lacking recently found fusion-indispensable genes, Fimp, Sof1, Spaca6, or Tmem95. Collectively, our data suggest that IZUMO1 is required for the sperm-oolemma binding prior to fusion at least in rat.


eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Xi Cao ◽  
Tingting Shi ◽  
Chuanhai Zhang ◽  
Wanzhu Jin ◽  
Lini Song ◽  
...  

Identification of key regulators of energy homeostasis holds important therapeutic promise for metabolic disorders, such as obesity and diabetes. ACE2 cleaves angiotensin II (Ang II) to generate Ang-(1-7) which acts mainly through the Mas1 receptor. Here, we identify ACE2 pathway as a critical regulator in the maintenance of thermogenesis and energy expenditure. We found that ACE2 is highly expressed in brown adipose tissue (BAT) and that cold stimulation increases ACE2 and Ang-(1-7) levels in BAT and serum. Ace2 knockout mice (Ace2-/y) and Mas1 knockout mice (Mas1-/-) displayed impaired thermogenesis. Mice transplanted with brown adipose tissue from Mas1-/- display metabolic abnormalities consistent with those seen in the Ace2 and Mas1 knockout mice. In contrast, impaired thermogenesis of Leprdb/db obese diabetic mice and high-fat diet-induced obese mice were ameliorated by overexpression of Ace2 or continuous infusion of Ang-(1-7). Activation of ACE2 pathway was associated with improvement of metabolic parameters, including blood glucose, lipids and energy expenditure in multiple animal models. Consistently, ACE2 pathway remarkably enhanced the browning of white adipose tissue. Mechanistically, we showed that ACE2 pathway activated Akt/FoxO1 and PKA pathway, leading to induction of UCP1 and activation of mitochondrial function. Our data propose that adaptive thermogenesis requires regulation of ACE2 pathway and highlight novel potential therapeutic targets for the treatment of metabolic disorders.


2022 ◽  
Vol 23 (2) ◽  
pp. 740
Author(s):  
Jocelyne Magré ◽  
Xavier Prieur

Obesity prevalence is increasing worldwide, leading to cardiometabolic morbidities. Adipocyte dysfunction, impairing white adipose tissue (WAT) expandability and metabolic flexibility, is central in the development of obesity-related metabolic complications. Rare syndromes of lipodystrophy characterized by an extreme paucity of functional adipose tissue should be considered as primary adipocyte dysfunction diseases. Berardinelli-Seip congenital lipodystrophy (BSCL) is the most severe form with a near absence of WAT associated with cardiometabolic complications such as insulin resistance, liver steatosis, dyslipidemia, and cardiomyopathy. Twenty years ago, mutations in the BSCL2 gene have been identified as the cause of BSCL in human. BSCL2 encodes seipin, an endoplasmic reticulum (ER) anchored protein whose function was unknown back then. Studies of seipin knockout mice or rats demonstrated how seipin deficiency leads to severe lipodystrophy and to cardiometabolic complications. At the cellular levels, seipin is organized in multimers that are particularly enriched at ER/lipid droplet and ER/mitochondria contact sites. Seipin deficiency impairs both adipocyte differentiation and mature adipocyte maintenance. Experiments using adipose tissue transplantation in seipin knockout mice and tissue-specific deletion of seipin have provided a large body of evidence that liver steatosis, cardiomyopathy, and renal injury, classical diabetic complications, are all consequences of lipodystrophy. Rare adipocyte dysfunctions such as in BSCL are the key paradigm to unravel the pathways that control adipocyte homeostasis. The knowledge gathered through the study of these pathologies may bring new strategies to maintain and improve adipose tissue expandability.


2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Laura M. de Jong ◽  
Zhengzheng Zhang ◽  
Yvette den Hartog ◽  
Timothy J. P. Sijsenaar ◽  
Renata Martins Cardoso ◽  
...  

AbstractProtein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled.


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