scholarly journals Critical roles for murine Reck in the regulation of vascular patterning and stabilization

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Glícia Maria de Almeida ◽  
Mako Yamamoto ◽  
Yoko Morioka ◽  
Shuichiro Ogawa ◽  
Tomoko Matsuzaki ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conditional Knockout ◽  
Vascular Patterning ◽  
Sprouting Angiogenesis ◽  
Mural Cells ◽  
Mammalian Embryogenesis ◽  
Underlying Causes ◽  
Aortic Explants ◽  
Tip Cells ◽  
Correct Association

Abstract Extracellular matrix (ECM) is known to play several important roles in vascular development, although the molecular mechanisms behind these remain largely unknown. RECK, a tumor suppressor downregulated in a wide variety of cancers, encodes a membrane-anchored matrix-metalloproteinase-regulator. Mice lacking functional Reck die in utero, demonstrating its importance for mammalian embryogenesis; however, the underlying causes of mid-gestation lethality remain unclear. Using Reck conditional knockout mice, we have now demonstrated that the lack of Reck in vascular mural cells is largely responsible for mid-gestation lethality. Experiments using cultured aortic explants further revealed that Reck is essential for at least two events in sprouting angiogenesis; (1) correct association of mural and endothelial tip cells to the microvessels and (2) maintenance of fibronectin matrix surrounding the vessels. These findings demonstrate the importance of appropriate cell-cell interactions and ECM maintenance for angiogenesis and the involvement of Reck as a critical regulator of these events.

Abstract 041: mTORC1 is Required for Leptin-induced Sympathetic Activation to the Kidney but Not Brown Adipose Tissue

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Balyssa B Bell ◽  
Donald A Morgan ◽  
Kamal Rahmouni
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Molecular Mechanisms ◽  
Leptin Receptor ◽  
Critical Role ◽  
Conditional Knockout ◽  
Sympathetic Activation ◽  
Long Term Effects ◽  
Brown Adipose ◽  
Mtorc1 Signaling

The adipocyte-derived hormone leptin plays a critical role in the regulation of energy homeostasis through its action in the brain to decrease food intake and promote energy expenditure by increasing sympathetic nerve activity (SNA) to the thermogenic brown adipose tissue (BAT). Leptin also increases SNA to cardiovascular organs including the kidney and raises arterial pressure. However, it is unclear whether leptin controls regional SNA via conserved or distinct molecular mechanisms. Multiple intracellular pathways have been associated with leptin signaling including the mechanistic target of rapamycin complex 1 (mTORC1), which has been proposed as a critical determinant of leptin action. Here, we assessed the contribution of mTORC1 signaling to leptin-evoked regional sympathetic activation. Simultaneous multifiber recording of renal and BAT SNA in anesthetized C57BL/6J mice showed that intracerebroventricular (ICV) administration of leptin (2μg, n=5) increased both renal (170±34%) and BAT (208±37%) SNA. Interestingly, ICV pre-treatment with the mTORC1 inhibitor (rapamycin, 5ng, n=6) abolished the leptin-induced increase in renal (10±6%, P<0.05 vs controls) but not BAT (226±31%) SNA. Next, we used conditional knockout mice that lack the critical mTORC1 subunit, Raptor, specifically in leptin receptor (LRb)-expressing cells (LRb Cre /Raptor fl/fl ) to determine the long-term effects of disrupting mTORC1 signaling on leptin-evoked increase in regional SNA. We confirmed the inability of leptin to activate mTORC1 signaling in LRb-expressing cells of LRb Cre /Raptor fl/fl mice relative to controls using immunohistochemical staining of phosphorylated ribosomal S6, a downstream target of mTORC1. We observed a significant increase in renal SNA in response to ICV leptin in control mice (127±16%, n=9), but not in LRb Cre /Raptor fl/fl mice (-4±15%, n=9, P<0.05 vs controls). Conversely, ICV leptin-induced increase in BAT SNA was not different in LRb Cre /Raptor fl/fl mice (109±27%, n=5) vs. littermate controls (173±52%, n=4). Our data suggest a critical role for mTORC1 signaling in selectively mediating the cardiovascular sympathetic but not the thermogenic actions of leptin, with important implications for obesity-associated hypertension.

Abstract 15907: Disruption of HCN2 Leads to Laterality Defects and Cardiac Arrhythmia

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Bin Ye ◽  
Greg Taycher ◽  
Tim Hacker ◽  
Leslie Chan ◽  
Nian-Qing Shi
Keyword(s):  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Heart Defects ◽  
Cell Types ◽  
Conditional Knockout ◽  
Hcn Channels ◽  
Special Diet ◽  
Ultrasound Study ◽  
Central Nervous Systems ◽  
Laterality Defects

Background: Congenital heart defects (CHDs) encompass a large numbers of cardiovascular malformations, and remain the major cause of infant mortality among all types of birth defects. However, molecular mechanisms underlying CHDs remain elusive, largely owning to the complexity of the diseases and lack of animal models that can reproduce the pathophysiological conditions in a laboratory setting. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN) are responsible for generating spontaneous pacemaker activities in cardiac and central nervous systems. These channels are also detected in other cell types such as ventricular myocytes. HCN currents recorded from neonatal cells have an activation threshold of -70 mV while those recorded from adult cells are activated at -110 mV. This difference indicates that HCN activity might be important in early development. However, roles of HCN channels in cardiogenesis and development are not fully understood. Methods and Results: We created a HCN2 conditional knockout (HCN2KO) model in which the full-length HCN2 was disrupted. Two KO lines were subsequently derived from this model. The first line was weaned by 21 days and they all died by 4-5 wks of age. Maternal ultrasound study revealed that these KO mice developed fetal arrhythmia and had an underdeveloped left side in their hearts. The second line was able to live on under our special diet/care. These mice displayed a slower growth rate (1.1±0.2 g/wk) and lower body weight (15.1±2.0 g) relative to their age-matched WT controls (2.2±0.1 g/wk and 27.5±1.5 g; n=9-10, p<0.05). Echocardiography and tissue staining data suggested that KO hearts had laterality defects compared to their size-matched WT controls. The survived KO mice were able to maintain cardiac function by developing much thicker anterior and posterior walls to sustain blood-pumping (n=6, p<0.05). Electrocardiographic results indicated that the average heart rate recorded from KO mice was ~100 bpm slower than their age-matched WT controls (n=5-6, p<0.05). Conclusions: These novel findings indicate that HCN2 is indispensable in mouse cardiogenesis and development. Our KO models are therefore innovative platforms for future CHD research.

Abstract 3640: Mitochondrial Uncoupling Protein 2 Facilitates Endothelial Cell Growth and Angiogenesis via Reduction of Mitochondrial Superoxide

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yukio Shimasaki ◽  
Kai Chen ◽  
John F Keaney
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Growth ◽  
Mitochondrial Function ◽  
Endothelial Cell Proliferation ◽  
Wild Type ◽  
Sprouting Angiogenesis ◽  
Mitochondrial Superoxide ◽  
Endothelial Cell Growth ◽  
Aortic Explants

Background: Growing evidence suggests that mitochondrial function contributes to cell phenotype. One important component of mitochondrial function is the membrane potential that is controlled, in part, by uncoupling proteins (UCPs). Based on our previous data, the UCP2 is predominantly expressed in cultured endothelial cells. Therefore, we sought to examine the role of UCP2 in endothelial cell growth and angiogenesis. Methods and Results: Murine lung endothelial cells (MLECs) were isolated from UCP2-null and wild-type mice. UCP2-null cells were found less proliferative than wild-type cells (P<0.02, UCP2-null cells vs. wild-type cells, n=4). This defect of UCP2-null cells was rescued by UCP2 adenovirus transfection (19% increase, p<0.02 vs. LacZ adenovirus treated cells, n=3), and also rescued by transfection with manganese superoxide dismutase (MnSOD) adenovirus (53% increase, P<0.002 vs. LacZ adenovirus treated cells, n=3). We found a reciprocal relation such as no UCP2 expression and higher mitochondrial superoxide level in the MLECs (P<0.005, UCP2-null cells vs. wild-type cells, n=3), suggesting that mitochondrial superoxide may regulate endothelial cell growth. Then, we prepared murine aortic rings from UCP2-null and wild-type mice and embedded in rat tail collagen gel. The sprouting angiogenesis of UCP2-null explants was significantly less than wild-type explants (P<0.02, UCP2-null explants vs. wild-type explants, n=3– 4). Furthermore, MLECs from MnSOD-heterozygous mice showed less proliferation with lower expression of UCP2 protein and higher mitochondrial superoxide level compared to the MLECs from wild-type littermates (P<0.02, MnSOD-heterozygous cells vs. wild-type cells, n=4 – 8). We also observed less sprouting angiogenesis in MnSOD-heterozygous aortic explants than wild-type aortic explants (P<0.05, MnSOD-heterozygous explants vs. wild-type explants, n=3– 6). Conclusions: These data indicate that mitochondrial superoxide controls endothelial cell proliferation and angiogenesis, suggesting that mitochondrial metabolism modulates the endothelial cell growth and angiogenesis.

scholarly journals HID-1 is required for homotypic fusion of immature secretory granules during maturation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Wen Du ◽  
Maoge Zhou ◽  
Wei Zhao ◽  
Dongwan Cheng ◽  
Lifen Wang ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Three Dimensional ◽  
Conditional Knockout ◽  
Active Peptides ◽  
Early Maturation ◽  
Proinsulin Processing ◽  
Granule Maturation ◽  
Golgi Network

Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules.

scholarly journals Ablation of the MiR-17-92 MicroRNA Cluster in Germ Cells Causes Subfertility in Female Mice

2018 ◽  
Vol 45 (2) ◽  
pp. 491-504 ◽  
Author(s):  
Jian Wang ◽  
Bo Xu ◽  
Geng G. Tian ◽  
Tao Sun ◽  
Ji Wu
Keyword(s):  
Germ Cells ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Conditional Knockout ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Follicular Atresia ◽  
Apoptotic Pathway ◽  
Female Mice ◽  
Important Regulator

Background/Aims: Oogenesis is a highly complex process that is intricately regulated by interactions of multiple genes and signaling molecules. However, the underlying molecular mechanisms are poorly understood. There is emerging evidence that microRNAs contribute to oogenesis. Here, we aimed to investigate the role of miR-17-92 cluster in regulating oogenesis. Methods: The miR-17-92 cluster was genetically ablated in germ cells of female mice by applying the Cre-loxp system for conditional gene knockout. Mating experiment, superovulation and histological analysis were used to assess the fertility of the model female mice. TUNEL assay was used to identify apoptotic cells in ovaries. The expression level of apoptosis- and follicular atresia- related genes was evaluated by qRT-PCR. Western blotting was performed to detect protein expression. Bioinformatics software and dual luciferase reporter assay were applied to predict and verify the target of miR-17-92 cluster. Results: Deletion of miR-17-92 cluster in germ cells of female mice caused increased oocyte degradation and follicular atresia, perturbed oogenesis, and ultimately led to subfertility. Genes involved in follicular atresia and the mitochondrial apoptotic pathway were obviously up-regulated. Furthermore, we verified that miR-19a regulated oogenesis at the post-transcriptional level by targeting Bmf in the ovaries of miR-17-92 cluster conditional knockout female mice. Conclusion: The miR-17-92 cluster is an important regulator of oogenesis. These findings will assist in better understanding the etiology of disorders in oogenesis and in developing new therapeutic targets for female infertility.

The histone acetyltransferase HBO1 (KAT7) promotes efficient tip cell sprouting during angiogenesis

Development ◽  
2021 ◽  
Author(s):  
Zoe L. Grant ◽  
Peter F. Hickey ◽  
Waruni Abeysekera ◽  
Lachlan Whitehead ◽  
Sabrina M. Lewis ◽  
...  
Keyword(s):  
Histone Acetyltransferase ◽  
Dynamic Changes ◽  
Cell Behaviour ◽  
Tip Cell ◽  
Sprouting Angiogenesis ◽  
A Genome ◽  
Vessel Growth ◽  
Intergenic Regions ◽  
Tip Cells

Blood vessel growth and remodelling are essential during embryonic development and disease pathogenesis. The diversity of endothelial cells (ECs) is transcriptionally evident and ECs undergo dynamic changes in gene expression during vessel growth and remodelling. Here, we investigated the role of the histone acetyltransferase HBO1 (KAT7), which is important for activating genes during development and histone H3 lysine 14 acetylation (H3K14ac). Loss of HBO1 and H3K14ac impaired developmental sprouting angiogenesis and reduced pathological EC overgrowth in the retinal endothelium. Single-cell RNA-sequencing of retinal ECs revealed an increased abundance of tip cells in Hbo1 deleted retinas, which lead to EC overcrowding in the retinal sprouting front and prevented efficient tip cell migration. We found that H3K14ac was highly abundant in the endothelial genome in both intra- and intergenic regions suggesting that the role of HBO1 is as a genome organiser that promotes efficient tip cell behaviour necessary for sprouting angiogenesis.

scholarly journals TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

2020 ◽  
Vol 21 (8) ◽  
pp. 2735 ◽  
Author(s):  
Enaam Chleilat ◽  
Abhishek Pethe ◽  
Dietmar Pfeifer ◽  
Kerstin Krieglstein ◽  
Eleni Roussa
Keyword(s):  
Oxidative Stress ◽  
Transforming Growth Factor Beta ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Cell Function ◽  
Dorsal Raphe ◽  
Conditional Knockout ◽  
Serotonergic Neurons ◽  
Dorsal Raphé

Calcium homeostasis is a cellular process required for proper cell function and survival, maintained by the coordinated action of several transporters, among them members of the Na+/Ca2+-exchanger family, such as SLC8A3. Transforming growth factor beta (TGF-β) signaling defines neuronal development and survival and may regulate the expression of channels and transporters. We investigated the regulation of SLC8A3 by TGF-β in a conditional knockout mouse with deletion of TGF-β signaling from Engrailed 1-expressing cells, i.e., in cells from the midbrain and rhombomere 1, and elucidated the underlying molecular mechanisms. The results show that SLC8A3 is significantly downregulated in developing dopaminergic and dorsal raphe serotonergic neurons in mutants and that low SLC8A3 abundance prevents the expression of the anti-apoptotic protein Bcl-xL. TGF-β signaling affects SLC8A3 via the canonical and p38 signaling pathway and may increase the binding of Smad4 to the Slc8a3 promoter. Expression of the lipid peroxidation marker malondialdehyde (MDA) was increased following knockdown of Slc8a3 expression in vitro. In neurons lacking TGF-β signaling, the number of MDA- and 4-hydroxynonenal (4-HNE)-positive cells was significantly increased, accompanied with increased cellular 4-HNE abundance. These results suggest that TGF-β contributes to the regulation of SLC8A3 expression in developing dopaminergic and dorsal raphe serotonergic neurons, thereby preventing oxidative stress.

scholarly journals APC/CCdh1-Rock2 pathway controls dendritic integrity and memory

2017 ◽  
Vol 114 (17) ◽  
pp. 4513-4518 ◽  
Author(s):  
Verónica Bobo-Jiménez ◽  
María Delgado-Esteban ◽  
Julie Angibaud ◽  
Irene Sánchez-Morán ◽  
Antonio de la Fuente ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Memory Loss ◽  
Neuronal Morphology ◽  
Conditional Knockout ◽  
Anaphase Promoting Complex ◽  
Adult Mice ◽  
Dendritic Network ◽  
Rho Protein ◽  
With Memory

Disruption of neuronal morphology contributes to the pathology of neurodegenerative disorders such as Alzheimer’s disease (AD). However, the underlying molecular mechanisms are unknown. Here, we show that postnatal deletion of Cdh1, a cofactor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase in neurons [Cdh1 conditional knockout (cKO)], disrupts dendrite arborization and causes dendritic spine and synapse loss in the cortex and hippocampus, concomitant with memory impairment and neurodegeneration, in adult mice. We found that the dendrite destabilizer Rho protein kinase 2 (Rock2), which accumulates in the brain of AD patients, is an APC/CCdh1 substrate in vivo and that Rock2 protein and activity increased in the cortex and hippocampus of Cdh1 cKO mice. In these animals, inhibition of Rock activity, using the clinically approved drug fasudil, prevented dendritic network disorganization, memory loss, and neurodegeneration. Thus, APC/CCdh1-mediated degradation of Rock2 maintains the dendritic network, memory formation, and neuronal survival, suggesting that pharmacological inhibition of aberrantly accumulated Rock2 may be a suitable therapeutic strategy against neurodegeneration.

Sprouting angiogenesis in human midterm uterus and fallopian tube is guided by endothelial tip cells

2012 ◽  
Vol 88 (1) ◽  
pp. 25-30 ◽  
Author(s):  
M. C. Rusu ◽  
A. G. M. Motoc ◽  
F. Pop ◽  
R. Folescu
Keyword(s):  
Fallopian Tube ◽  
Sprouting Angiogenesis ◽  
Tip Cells

scholarly journals Follistatin is critical for mouse uterine receptivity and decidualization

2017 ◽  
Vol 114 (24) ◽  
pp. E4772-E4781 ◽  
Author(s):  
Paul T. Fullerton ◽  
Diana Monsivais ◽  
Ramakrishna Kommagani ◽  
Martin M. Matzuk
Keyword(s):  
Molecular Mechanisms ◽  
Recurrent Miscarriage ◽  
Embryo Implantation ◽  
Bmp Signaling ◽  
Conditional Knockout ◽  
Uterine Receptivity ◽  
Vitro Fertilization ◽  
Morphogenetic Protein ◽  
Proliferation And Differentiation

Embryo implantation remains a significant challenge for assisted reproductive technology, with implantation failure occurring in ∼50% of in vitro fertilization attempts. Understanding the molecular mechanisms underlying uterine receptivity will enable the development of new interventions and biomarkers. TGFβ family signaling in the uterus is critical for establishing and maintaining pregnancy. Follistatin (FST) regulates TGFβ family signaling by selectively binding TGFβ family ligands and sequestering them. In humans, FST is up-regulated in the decidua during early pregnancy, and women with recurrent miscarriage have lower endometrial expression of FST during the luteal phase. Because global knockout of Fst is perinatal lethal in mice, we generated a conditional knockout (cKO) of Fst in the uterus using progesterone receptor-cre to study the roles of uterine Fst during pregnancy. Uterine Fst-cKO mice demonstrate severe fertility defects and deliver only 2% of the number of pups delivered by control females. In Fst-cKO mice, the uterine luminal epithelium does not respond properly to estrogen and progesterone signals and remains unreceptive to embryo attachment by continuing to proliferate and failing to differentiate. The uterine stroma of Fst-cKO mice also responds poorly to artificial decidualization, with lower levels of proliferation and differentiation. In the absence of uterine FST, activin B expression and signaling are up-regulated, and bone morphogenetic protein (BMP) signals are impaired. Our findings support a model in which repression of activin signaling by FST enables uterine receptivity by preserving critical BMP signaling.

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