Regulation of apoptosis in the endocardial cushions of the developing chick heart

2002 ◽  
Vol 282 (6) ◽  
pp. C1348-C1360 ◽  
Author(s):  
William M. Keyes ◽  
Esmond J. Sanders
Keyword(s):  
Heart Development ◽  
Time Course ◽  
Nuclear Staining ◽  
Caspase 9 ◽  
Endocardial Cushions ◽  
Similar Time ◽  
Cleavage Fragment ◽  
Protein Levels ◽  
Developing Heart

During the early stages of heart development, there are two main foci of cell death: outflow tract (OT) and atrioventricular (AV) endocardial cushions. These tissues contribute to the septa and valves of the mature heart and receive cell populations from neural crest (NC) cell migration and epicardial cell invasion. We examined embryonic chick hearts for expression, in the cushions, of bcl-2 family members, caspase-9, and the caspase substrate poly(ADP-ribose) polymerase. Antiapoptotic bcl-2 is expressed heavily in the OT and AV regions throughout embryonic days (ED) 4–7, with a decrease in levels at ED 4 and 5 in OT and AV cushions, respectively. Proapoptotic bax predominantly associated with the prongs of the NC-derived aorticopulmonary (AP) septum but was expressed throughout the AV cushions. Proapoptotic bak also associated with the prongs of the AP septum in the OT, while protein levels were upregulated at ED 4–5 and 4–6 in OT and AV cushions, respectively. Bid expression showed a similar time course. We found the 10-kDa cleavage fragment of active caspase-9 at ED 4–8 and 5–8 in OT and AV cushions, respectively, and the 24-kDa cleavage fragment of poly(ADP-ribose) polymerase throughout ED 3–8 and 7–8 in OT and AV cushions, respectively. Caspase-3 cleavage occurred throughout the time period examined. Using cushion cell cultures, we found that inhibitors of caspases-3 and -9 and a universal caspase inhibitor significantly reduced apoptosis, as did retroviral overexpression of bcl-2 using an RCAS expression vector. Premigratory NC cells were fluorescently labeled in vivo with 1,1-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine. Subsequent nuclear staining of cushion cells with 4,6-diamidino-2-phenylindole revealed the presence of apoptotic nuclei in the NC cells in the OT cushions and in the prongs of the AP septum. These results demonstrate a developmentally regulated role for the bcl-2 and the caspase families of molecules in the endocardial cushions of the developing heart and lend support to the possibility that some of the dying cells in the cushions are derived from the NC.

scholarly journals Insights to Heart Development and Cardiac Disease Models Using Pluripotent Stem Cell Derived 3D Organoids

2021 ◽  
Vol 9 ◽  
Author(s):  
Jeremy Kah Sheng Pang ◽  
Beatrice Xuan Ho ◽  
Woon-Khiong Chan ◽  
Boon-Seng Soh
Keyword(s):  
Cardiac Disease ◽  
Heart Development ◽  
Pluripotent Stem Cell ◽  
3D Models ◽  
Cardiac Diseases ◽  
Significant Progress ◽  
Fundamental Parameters ◽  
Developing Heart

Medical research in the recent years has achieved significant progress due to the increasing prominence of organoid technology. Various developed tissue organoids bridge the limitations of conventional 2D cell culture and animal models by recapitulating in vivo cellular complexity. Current 3D cardiac organoid cultures have shown their utility in modelling key developmental hallmarks of heart organogenesis, but the complexity of the organ demands a more versatile model that can investigate more fundamental parameters, such as structure, organization and compartmentalization of a functioning heart. This review will cover the prominence of cardiac organoids in recent research, unpack current in vitro 3D models of the developing heart and look into the prospect of developing physiologically appropriate cardiac organoids with translational applicability. In addition, we discuss some of the limitations of existing cardiac organoid models in modelling embryonic development of the heart and manifestation of cardiac diseases.

In vivo study on medullary H(+)-sensitive neurons

1990 ◽  
Vol 69 (4) ◽  
pp. 1408-1412 ◽  
Author(s):  
N. Kogo ◽  
H. Arita
Keyword(s):  
Time Course ◽  
Direct Application ◽  
Ventrolateral Medulla ◽  
Similar Time ◽  
Central Chemoreceptors ◽  
Ph Dependent ◽  
Spontaneously Breathing ◽  
Medullary Neurons ◽  
Stimulation Of

Using the micro pressure ejection technique, we examined responses of medullary neurons with nonphasic discharges (164 units) to direct application of acidified mock cerebrospinal fluid (CSF, pH 6.85-7.05) in decerebrated spontaneously breathing cats. We found 16 H(+)-sensitive cells; they were excited promptly on application of approximately 500 pl of acidified mock CSF in the vicinity of the neuron under investigation, whereas they were unaffected by microejection of the control mock CSF (pH 7.25-7.60). Of the 16 H(+)-sensitive cells, 10 units were further found to be excited by transcapillary stimulation of the central chemoreceptors by using a method of intravertebral arterial injection of CO2-saturated saline. The discharges increased in a similar time course to that of ventilatory augmentation. Distributions of these 10 specific H(+)-sensitive cells were found in the vicinity of nucleus tractus solitarii as well as deep in the ventrolateral medulla. The present results suggest a possibility that pH-dependent central chemoreceptors, if any, would be located in two distinct medullary regions described in this study.

scholarly journals β-Catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse

2004 ◽  
Vol 166 (3) ◽  
pp. 359-367 ◽  
Author(s):  
Stefan Liebner ◽  
Anna Cattelino ◽  
Radiosa Gallini ◽  
Noemi Rudini ◽  
Monica Iurlaro ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Heart Development ◽  
Transcriptional Activity ◽  
Ex Vivo ◽  
Smooth Muscle Actin ◽  
Muscle Actin ◽  
Endocardial Cushions ◽  
Smad Phosphorylation ◽  
Mesenchymal Transformation

During heart development endocardial cells within the atrio-ventricular (AV) region undergo TGFβ-dependent epithelial-mesenchymal transformation (EMT) and invade the underlying cardiac jelly. This process gives rise to the endocardial cushions from which AV valves and part of the septum originate. In this paper we show that in mouse embryos and in AV explants TGFβ induction of endocardial EMT is strongly inhibited in mice deficient for endothelial β-catenin, leading to a lack of heart cushion formation. Using a Wnt-signaling reporter mouse strain, we demonstrated in vivo and ex vivo that EMT in heart cushion is accompanied by activation of β-catenin/TCF/Lef transcriptional activity. In cultured endothelial cells, TGFβ2 induces α-smooth muscle actin (αSMA) expression. This process was strongly reduced in β-catenin null cells, although TGFβ2 induced smad phosphorylation was unchanged. These data demonstrate an involvement of β-catenin/TCF/Lef transcriptional activity in heart cushion formation, and suggest an interaction between TGFβ and Wnt-signaling pathways in the induction of endothelial-mesenchymal transformation.

scholarly journals Mutation in the Trapα/Ssr1 Gene, Encoding Translocon-Associated Protein α, Results in Outflow Tract Morphogenetic Defects

2006 ◽  
Vol 26 (20) ◽  
pp. 7760-7771 ◽  
Author(s):  
K. Mesbah ◽  
A. Camus ◽  
C. Babinet ◽  
J. Barra
Keyword(s):  
Heart Development ◽  
Proximal Part ◽  
Double Outlet Right Ventricle ◽  
Outflow Tract ◽  
Endoplasmic Reticulum Membrane ◽  
Messenger Rnas ◽  
Endocardial Cushions ◽  
Gene Encoding ◽  
Mutant Cells

ABSTRACT Translocon-associated protein complex (TRAP) is thought to be required for efficient protein-specific translocation across the endoplasmic reticulum membrane. We created a mutation in the Trapα gene that leads to the synthesis of a truncated TRAPα protein fused to ShBle-β-galactosidase. Analysis of Trapα cDNAs reveals that among three different messenger RNAs expressed in the mouse, one of them encodes a slightly larger protein that differs in its C-terminal end. This mRNA, specific for skeletal muscle and heart, is only expressed after birth. Homozygous Trapα mutant pups die at birth, likely as a result of severe cardiac defects. Indeed, the septation of the proximal part of the outflow tract is absent, resulting in a double-outlet right ventricle. Studies of protein secretion in transfected embryonic fibroblasts reveal that the TRAP complex does not function properly in homozygous mutant cells and confirm, in vivo, the involvement of TRAP in substrate-specific translocation. Our results provide the first in vivo demonstration that a member of the TRAP complex plays a crucial role in mammalian heart development and suggest that TRAPα could be involved in translocation of factors necessary for maturation of endocardial cushions.

scholarly journals Temporal and Spatial Profile of Bid Cleavage after Experimental Traumatic Brain Injury

2002 ◽  
Vol 22 (8) ◽  
pp. 951-958 ◽  
Author(s):  
Gerhard Franz ◽  
Ronny Beer ◽  
Denis Intemann ◽  
Stanislaw Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Brain Injury ◽  
Brain Regions ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Cell Degeneration ◽  
Similar Time ◽  
Impact Injury ◽  
Experimental Traumatic Brain Injury

Apoptosis plays an essential role in the cascade of CNS cell degeneration after traumatic brain injury. However, the underlying mechanisms are poorly understood. The authors examined the temporal profile and cell subtype distribution of the proapoptotic protein Bid from 6 hours to 7 days after cortical impact injury in the rat. Increased protein levels of tBid were seen in the cortex ipsilateral to the injury site from 6 hours to 3 days after trauma. Immunohistologic examinations revealed expression of tBid in neurons, astrocytes, and oligodendrocytes from 6 hours to 3 days after impact injury, and concurrent assessment of DNA damage using TUNEL identified tBid-immunopositive cells with apoptoticlike morphology in the traumatized cortex. Moreover, Bid cleavage and activation of caspase-8 and caspase-9 occurred at similar time points and in similar brain regions (i.e., cortical layers 2 to 5) after impact injury. In contrast, there was no evidence of caspase-8 or caspase-9 processing or Bid cleavage in the ipsilateral hippocampus, contralateral cortex, and hippocampus up to 7 days after the injury. The results provide the first evidence of Bid cleavage in the traumatized cortex after experimental traumatic brain injury in vivo, and demonstrate that tBid is expressed in neurons and glial cells. Further, findings indicate that cleavage of Bid may be associated with the activation of the initiator caspase-8 and caspase-9. Finally, these data support the hypothesis that cleavage of Bid contributes to the apoptotic degeneration of different CNS cells in the injured cortex.

scholarly journals Analysis of cardiac differentiation at single cell resolution reveals a requirement of hypertrophic signaling for HOPX transcription

2017 ◽  
Author(s):  
Clayton E Friedman ◽  
Quan Nguyen ◽  
Samuel W Lukowski ◽  
Han Sheng Chiu ◽  
Abbigail Helfer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Heart Development ◽  
Time Course ◽  
Cardiac Differentiation ◽  
Transcriptional Networks ◽  
Hypertrophic Signaling

AbstractDifferentiation into diverse cell lineages requires the orchestration of gene regulatory networks guiding diverse cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from 43,168 cells across five time points over a thirty day time-course of in vitro cardiac-directed differentiation. Unsupervised clustering and lineage prediction algorithms were used to map fate choices and transcriptional networks underlying cardiac differentiation. We leveraged this resource to identify strategies for controlling in vitro differentiation as it occurs in vivo. HOPX, a non-DNA binding homeodomain protein essential for heart development in vivo was identified as dys-regulated in in vitro derived cardiomyocytes. Utilizing genetic gain and loss of function approaches, we dissect the transcriptional complexity of the HOPX locus and identify the requirement of hypertrophic signaling for HOPX transcription in hPSC-derived cardiomyocytes. This work provides a single cell dissection of the transcriptional landscape of cardiac differentiation for broad applications of stem cells in cardiovascular biology.

Effects of a high-glucose environment on the pituitary growth hormone-releasing hormone receptor: type 1 diabetes compared with in vitro glucotoxicity

2008 ◽  
Vol 294 (4) ◽  
pp. E740-E751 ◽  
Author(s):  
Karine Bédard ◽  
Julie Strecko ◽  
Karyne Thériault ◽  
Julie Bédard ◽  
Christelle Veyrat-Durebex ◽  
...  
Keyword(s):  
High Glucose ◽  
Time Course ◽  
Diabetic Rats ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Mrna Transcript ◽  
Protein Levels ◽  
Igf I

The present study investigated the effects of diabetes and high glucose on GHRH receptor (GHRH-R) mRNA and protein levels in the pituitary of diabetic rats 2, 21, and 60 days post-streptozotocin (post-STZ) administration. Two days post-STZ, the 2.5-kb GHRH-R mRNA transcript was increased. Twenty-one days post-STZ, both the 2.5- and 4-kb transcripts and a 72-kDa 125I-GHRH-GHRH-R complex were elevated. Sixty days post-STZ, the 4-kb transcript remained increased and the 45-kDa 125I-GHRH-GHRH-R complex (functional receptor) was decreased. Hypothalamic GHRH mRNA and serum total IGF-I levels were reduced at all three time points. To better understand the role of high glucose on GHRH-R regulation, time-course effects of 33 compared with 6 mM d-glucose (DG) were examined in cultured anterior pituitary cells from 2-mo-old healthy rats. Membrane lipoperoxidation was present in 33 mM DG, and GHRH-R mRNA levels were diminished after 24 h, Fluo-GHRH internalization was marginal after 16–24 h, and GHRH-induced cAMP levels were decreased after 24 and 48 h. Altogether, these results indicate that the increase of the 2.5-kb GHRH-R mRNA transcript in vivo could be a consequence of a decrease of hypothalamic GHRH mRNA levels in STZ rats. Since it does not affect primarily functional GHRH-R levels, the initial diminution of circulating IGF-I levels could result from a decreased GHRH-R stimulation by GHRH. Thus, the effect of glucotoxicity would be related to a decrease of functional GHRH-R protein, as observed in rats 60 days post-STZ and in cultured pituitary cells from healthy rats exposed to a high-glucose environment.

Apoptotic myocytes generate monocyte chemoattractant protein-1 and mediate macrophage recruitment

2008 ◽  
Vol 104 (3) ◽  
pp. 601-609 ◽  
Author(s):  
Miyuki Kobara ◽  
Nahoko Sunagawa ◽  
Masaki Abe ◽  
Nana Tanaka ◽  
Hiroe Toba ◽  
...  
Keyword(s):  
Critical Role ◽  
Neonatal Rat ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Nuclear Staining ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Protein Levels ◽  
Monocyte Chemoattractant Protein

The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-1 (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2–1 μmol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 μmol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-1 mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-1. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a trans-well system. The culture medium of STS-treated myocytes recruited monocytes in a MCP-1-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 min of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 ± 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP-1 antibody showed that 69.8 ± 3.9% of TUNEL-positive myocytes expressed MCP-1 protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-1, which have a critical role in the active recruitment of macrophages.

A transgenic zebrafish model of neutrophilic inflammation

Blood ◽  
2006 ◽  
Vol 108 (13) ◽  
pp. 3976-3978 ◽  
Author(s):  
Stephen A. Renshaw ◽  
Catherine A. Loynes ◽  
Daniel M.I. Trushell ◽  
Stone Elworthy ◽  
Philip W. Ingham ◽  
...  
Keyword(s):  
Time Course ◽  
Digital Image Analysis ◽  
Experimental Manipulation ◽  
In Vivo Model ◽  
Transgenic Zebrafish ◽  
Zebrafish Model ◽  
Similar Time ◽  
Zebrafish Larvae ◽  
Fluorescent Cells

Abstract We have established an in vivo model for genetic analysis of the inflammatory response by generating a transgenic zebrafish line that expresses GFP under the neutrophil-specific myeloperoxidase promoter. We show that inflammation is induced after transection of the tail of zebrafish larvae and that this inflammation subsequently resolves over a similar time course to mammalian systems. Quantitative data can be generated from this model by counting of fluorescent cells or by digital image analysis. In addition, we show that the resolution of experimentally induced inflammation can be inhibited by the addition of a pancaspase inhibitor, zVD.fmk, demonstrating that experimental manipulation of the resolution of inflammation is possible in this model.

DER TSH-RESERVE-TEST AN DER RATTE

1963 ◽  
Vol 44 (4) ◽  
pp. 570-580 ◽  
Author(s):  
H. W. Iff ◽  
H. Studer ◽  
F. Wyss
Keyword(s):  
Time Course ◽  
Term Treatment ◽  
Short Term ◽  
Iodide Uptake ◽  
Short Term Treatment ◽  
Similar Time ◽  
Clinical Method ◽  
Tsh Secretion

ABSTRACT A rebound of 131I-uptake by the thyroid gland after a thyrostatic treatment may be taken as evidence of an unimpaired pituitary TSH-secretion. The iodide uptake in vivo and the iodide accumulation in vitro were studied in rat thyroids following a short-term treatment of the animals with carbimazole. The experiments served as models for the clinical method of assaying the pituitary TSH-reserve. The total iodide uptake reaches a peak 36 hours after the end of a carbimazole treatment and returns to normal after 96 hours. The rebound of the iodide accumulation has a similar time course. Extending the carbimazole treatment from 6 to 12 days leads to a definite increase in the peak iodide accumulation while the peak of the total iodide uptake was not significantly increased. The duration of the rebound-phase is not changed by prolonged carbimazole treatment.

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