scholarly journals A blood flow–dependent klf2a-NO signaling cascade is required for stabilization of hematopoietic stem cell programming in zebrafish embryos

Blood ◽  
2011 ◽  
Vol 118 (15) ◽  
pp. 4102-4110 ◽  
Author(s):  
Lu Wang ◽  
Panpan Zhang ◽  
Yonglong Wei ◽  
Ya Gao ◽  
Roger Patient ◽  
...  
Keyword(s):  
Blood Flow ◽  
Signaling Cascade ◽  
Zebrafish Embryos ◽  
Hematopoietic Stem ◽  
Hemodynamic Forces ◽  
No Signaling ◽  
Vessel Development ◽  
And Function ◽  
Chip Analysis

Abstract Blood flow has long been thought to be important for vessel development and function, but its role in HSC development is not yet fully understood. Here, we take advantage of zebrafish embryos with circulation defects that retain relatively normal early development to illustrate the combinatorial roles of genetic and hemodynamic forces in HSC development. We show that blood flow is not required for initiation of HSC gene expression, but instead is indispensable for its maintenance. Knockdown of klf2a mimics the silent heart (sih/tnnt2a) phenotype while overexpression of klf2a in tnnt2a morphant embryos can rescue HSC defects, suggesting that klf2a is a downstream mediator of blood flow. Furthermore, the expression of NO synthase (nos) was reduced in klf2a knockdown embryos, and ChIP analysis showed that endogenous Klf2a is bound to the promoters of nos genes in vivo, indicating direct gene regulation. Finally, administration of the NO agonist S-nitroso N-acetylpenicillamine (SNAP) can restore HSC development in tnnt2a and klf2a morphants, suggesting that NO signaling is downstream of Klf2a which is induced by hemodynamic forces. Taken together, we have demonstrated that blood flow is essential for HSC development and is mediated by a klf2a-NO signaling cascade in zebrafish.

Microarray profiling revealsCXCR4ais downregulated by blood flow in vivo and mediates collateral formation in zebrafish embryos

2009 ◽  
Vol 38 (3) ◽  
pp. 319-327 ◽  
Author(s):  
Ian M. Packham ◽  
Caroline Gray ◽  
Paul R. Heath ◽  
Paul G. Hellewell ◽  
Philip W. Ingham ◽  
...  
Keyword(s):  
Blood Flow ◽  
Cardiac Contraction ◽  
Zebrafish Embryos ◽  
Knock Down ◽  
Transcriptional Effect ◽  
Vessel Development ◽  
Hemodynamic Force ◽  
Collateral Vessels ◽  
And Control

The response to hemodynamic force is implicated in a number of pathologies including collateral vessel development. However, the transcriptional effect of hemodynamic force is extremely challenging to examine in vivo in mammals without also detecting confounding processes such as hypoxia and ischemia. We therefore serially examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since they obtain sufficient oxygenation by diffusion. Morpholino antisense knock-down of cardiac troponin T2 ( tnnt2) prevented cardiac contraction without affecting vascular development. Gene expression in whole embryo RNA from tnnt2 or control morphants at 36, 48, and 60 h postfertilization (hpf) was assessed using Affymetrix GeneChip Zebrafish Genome Arrays (>14,900 transcripts). We identified 308 differentially expressed genes between tnnt2 and control morphants. One such ( CXCR4a) was significantly more highly expressed in tnnt2 morphants at 48 and 60 hpf than controls. In situ hybridization localized CXCR4a upregulation to endothelium of both tnnt2 morphants and gridlock mutants (which have an occluded aorta preventing distal blood flow). This upregulation appears to be of functional significance as either CXCR4a knock-down or pharmacologic inhibition impaired the ability of gridlock mutants to recover blood flow via collateral vessels. We conclude absence of hemodynamic force induces endothelial CXCR4a upregulation that promotes recovery of blood flow.

Hematopoietic Stem Cell Development Is Dependent on Blood Flow and Nitric Oxide Signaling

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 728-728 ◽  
Author(s):  
Trista E. North ◽  
Wolfram Goessling ◽  
Marian Peeters ◽  
Pulin Li ◽  
Allegra M. Lord ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Blood Flow ◽  
Physiological Mechanism ◽  
No Production ◽  
Hematopoietic Stem ◽  
Nitric Oxide Signaling ◽  
No Signaling ◽  
Increased Blood Flow

Abstract During vertebrate embryogenesis, definitive hematopoietic stem cells (HSC) arise in the aorta-gonads-mesonephros (AGM). Based on the functional conservation of AGM hematopoiesis from fish to man, an evolutionary advantage for the production of stem cells within the aorta must exist. The identification of the signals that induce HSCs at this developmental stage is of significant interest. Through a chemical genetic screen in zebrafish, a diverse group of compounds that regulate blood flow were found to affect the production of runx1/cmyb+ HSCs. These compounds represented modulators of the adrenergic and renin/angiotensin pathways, and Ca+, Na+ and nitric oxide (NO) signaling. In general, we determined that compounds which increased blood flow enhanced HSC number, whereas chemicals that decreased blood flow diminished runx1/cmyb expression. The conserved physiological mechanism of action of each compound on the vasculature was confirmed in vivo by confocal microscopy of transgenic fli1:GFP reporter fish. In the zebrafish, the step-wise initiation of heartbeat, establishment of vigorous circulation and onset of definitive hematopoiesis in the aorta-gonad-mesonephros region (AGM) suggests that blood flow may trigger HSC formation. silent heart (sih) embryos that lack a heartbeat and fail to establish blood circulation exhibit severely reduced numbers of runx1+ HSCs in the AGM. Blood flow modifying agents primarily exerted their effects after the onset of the heartbeat (>24 hpf), however, only compounds that increase NO production (L-Arginine, S-nitroso-N-acetyl-penicillamine (SNAP)) could induce HSC formation prior to the initiation of circulation (5 somites to 22 hpf). Furthermore, SNAP rescued HSC production in sih mutant zebrafish, whereas other drugs that increased blood flow could not. Treatment with the NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), and morpholino-oligonucleotide (MO)-knockdown of nos1 (nnos/enos) blocked HSC development. Additionally, modulation of downstream components of the NO pathway affected HSC production in the zebrafish embryo. Together these data indicate that NO signaling is the downstream effector of blood flow on AGM HSC induction. To document that NO-mediated regulation of HSC formation was conserved across vertebrate species, we examined definitive HSC production in the murine AGM. Nos3 (eNos) was found to be expressed in the AGM endothelium and aortic hematopoietic clusters. Additionally, Nos3 expression specifically marks the population of HSCs with long-term adult bone marrow repopulating activity. Intrauterine NOS inhibition with L-NAME resulted in a lack of hematopoietic clusters in the AGM and a failure to generate transplantable hematopoietic progenitors. Our work provides a direct link between the initiation of circulation and the onset of AGM hematopoiesis, and identifies NO signaling as a conserved downstream regulator of HSC development. ^TEN and WG contributed equally to this work

scholarly journals METTL3-dependent N6-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium

2021 ◽  
Vol 118 (7) ◽  
pp. e2025070118
Author(s):  
Chian-Shiu Chien ◽  
Julie Yi-Shuan Li ◽  
Yueh Chien ◽  
Mong-Lien Wang ◽  
Aliaksandr A. Yarmishyn ◽  
...  
Keyword(s):  
Blood Flow ◽  
Endothelial Activation ◽  
Plaque Formation ◽  
Rna Modification ◽  
Branch Points ◽  
Monocyte Adhesion ◽  
Down Regulation ◽  
Hemodynamic Forces

Atherosclerosis is characterized by the plaque formation that restricts intraarterial blood flow. The disturbed blood flow with the associated oscillatory stress (OS) at the arterial curvatures and branch points can trigger endothelial activation and is one of the risk factors of atherosclerosis. Many studies reported the mechanotransduction related to OS and atherogenesis; however, the transcriptional and posttranscriptional regulatory mechanisms of atherosclerosis remain unclear. Herein, we investigated the role of N6-methyladenosine (m6A) RNA methylation in mechanotransduction in endothelial cells (ECs) because of its important role in epitranscriptome regulation. We have identified m6A methyltransferase METTL3 as a responsive hub to hemodynamic forces and atherogenic stimuli in ECs. OS led to an up-regulation of METTL3 expression, accompanied by m6A RNA hypermethylation, increased NF-κB p65 Ser536 phosphorylation, and enhanced monocyte adhesion. Knockdown of METTL3 abrogated this OS-induced m6A RNA hypermethylation and other manifestations, while METTL3 overexpression led to changes resembling the OS effects. RNA-sequencing and m6A-enhanced cross-linking and immunoprecipitation (eCLIP) experiments revealed NLRP1 and KLF4 as two hemodynamics-related downstream targets of METTL3-mediated hypermethylation. The METTL3-mediated RNA hypermethylation up-regulated NLRP1 transcript and down-regulated KLF4 transcript through YTHDF1 and YTHDF2 m6A reader proteins, respectively. In the in vivo atherosclerosis model, partial ligation of the carotid artery led to plaque formation and up-regulation of METTL3 and NLRP1, with down-regulation of KLF4; knockdown of METTL3 via repetitive shRNA administration prevented the atherogenic process, NLRP3 up-regulation, and KLF4 down-regulation. Collectively, we have demonstrated that METTL3 serves a central role in the atherogenesis induced by OS and disturbed blood flow.

Strontium can increase some osteoblasts without increasing hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1173-1181 ◽  
Author(s):  
Stefania Lymperi ◽  
Nicole Horwood ◽  
Stephen Marley ◽  
Myrtle Y. Gordon ◽  
Andrew P. Cope ◽  
...  
Keyword(s):  
Trabecular Thickness ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Parallel Change ◽  
Hsc Niche ◽  
And Function ◽  
Hematopoietic Niche ◽  
In Vitro Treatment

Abstract Osteoblasts are a key component in the regulation of the hematopoietic stem cell (HSC) niche. Manipulating osteoblast numbers results in a parallel change in HSC numbers. We tested the activity of strontium (Sr), a bone anabolic agent that enhances osteoblast function and inhibits osteoclast activity, on hematopoiesis. In vitro treatment of primary murine osteoblasts with Sr increased their ability to form bone nodules, and in vivo it increased osteoblast number, bone volume, and trabecular thickness and decreased trabecular pattern factor. However, the administration of Sr had no influence on primitive HSCs, although the number of hematopoietic progenitors was higher than in control cells. When Sr-treated mice were used as donors for HSC transplantation, no difference in the engraftment ability was observed, whereas hematopoietic recovery was delayed when they were used as recipients. Despite the changes in osteoblast numbers, no increment in the number of N-cadherin+ osteoblasts and N-cadherin transcripts could be detected in Sr-treated mice. Therefore, increasing the overall number and function of osteoblasts without increasing N-cadherin+ cells is not sufficient to enhance HSC quantity and function. Our study further supports the notion that N-cadherin+ osteoblasts are fundamental in the hematopoietic niche.

scholarly journals The chemokine GROβ mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 860-869 ◽  
Author(s):  
Seiji Fukuda ◽  
Huimin Bian ◽  
Andrew G. King ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
And Function ◽  
Stimulating Factor ◽  
Cxcr4 Axis

Abstract Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROβ (GROβΔ4/CXCL2Δ4) or the combination of GROβΔ4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROβ-mobilized grafts. PBSCs mobilized by GROβΔ4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage− (SKL) cells and more primitive CD34−-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROβΔ4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROβΔ4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1α in vitro that was associated with higher CD26 expression. However, GROβΔ4-mobilized SKL and c-Kit+ lineage− (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROβΔ4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROβΔ4-mobilized cells is less dependent on the SDF-1α/CXCR4 axis.

Temozolomide, irradiation and hypoxia promote homing of hematopoietic progenitor cells towards gliomas

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20044-20044
Author(s):  
W. Wick ◽  
G. Tabatabai ◽  
B. Frank ◽  
M. Weller
Keyword(s):  
Progenitor Cells ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Tumor Hypoxia ◽  
Glioma Cells ◽  
Signaling Cascade ◽  
Hematopoietic Stem ◽  
The Impact

20044 Background: Temozolomide and irradiation are essential parts of the standard therapy and hypoxia is a critical aspect of the microenvironment of gliomas. IN the present study, we aimed at investigating the impact of these stimuli on the previously defined transforming growth factor beta (TGF-β)- and stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1α/CXCL12)-dependent migration of adult hematopoietic stem and progenitor cells (HPC) towards glioma cells in vitro and the homing to experimental gliomas in vivo. Hyperthermia served as control. Methods and Results: Cerebral irradiation of nude mice at 21 days after intracerebral implantation of LNT-229 glioma induces tumor satellite formation and enhances the glioma tropism of HPC in vivo. Supernatants of temozolomide-treated, irradiated or hypoxic LNT-229 glioma cells promote HPC migration in vitro. Reporter assays reveal that the CXCL12 promoter activity is enhanced in LNT-229 glioma cells at 24 h after irradiation at 8 Gy or after exposure to 1% oxygen for 12 h. The irradiation- and hypoxia-induced release of CXCL12 depends on hypoxia inducible factor-1 alpha (HIF-1α), but not on p53. Induction of transcriptional activity of HIF-1α by hypoxia and irradiation requires an intact signaling cascade of TGF-β. Conclusions: Thus, we delineate a novel stress signaling cascade in glioma cells involving TGF-β, HIF-1α and CXCL12. Stress stimuli can be temozolomide, irradiation and hypoxia but not hyperthermia. These data suggest that the use of HPC as cellular vectors in the treatment of glioblastoma may well be combined with anti-angiogenic therapies which induce tumor hypoxia. [Table: see text]

scholarly journals In vivo single cell analysis reveals Gata2 dynamics in cells transitioning to hematopoietic fate

2017 ◽  
Vol 215 (1) ◽  
pp. 233-248 ◽  
Author(s):  
Christina Eich ◽  
Jochen Arlt ◽  
Chris S. Vink ◽  
Parham Solaimani Kartalaei ◽  
Polynikis Kaimakis ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Fate ◽  
Regulatory Networks ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Time Lapse ◽  
Hematopoietic Stem ◽  
And Function ◽  
In Cells

Cell fate is established through coordinated gene expression programs in individual cells. Regulatory networks that include the Gata2 transcription factor play central roles in hematopoietic fate establishment. Although Gata2 is essential to the embryonic development and function of hematopoietic stem cells that form the adult hierarchy, little is known about the in vivo expression dynamics of Gata2 in single cells. Here, we examine Gata2 expression in single aortic cells as they establish hematopoietic fate in Gata2Venus mouse embryos. Time-lapse imaging reveals rapid pulsatile level changes in Gata2 reporter expression in cells undergoing endothelial-to-hematopoietic transition. Moreover, Gata2 reporter pulsatile expression is dramatically altered in Gata2+/− aortic cells, which undergo fewer transitions and are reduced in hematopoietic potential. Our novel finding of dynamic pulsatile expression of Gata2 suggests a highly unstable genetic state in single cells concomitant with their transition to hematopoietic fate. This reinforces the notion that threshold levels of Gata2 influence fate establishment and has implications for transcription factor–related hematologic dysfunctions.

scholarly journals Expression and Function of Murine Receptor Tyrosine Kinases, TIE and TEK, in Hematopoietic Stem Cells

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4317-4326 ◽  
Author(s):  
Michihiro Yano ◽  
Atsushi Iwama ◽  
Hitoshi Nishio ◽  
Junko Suda ◽  
Goro Takada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Stem Cell Marker ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
And Function

Abstract Two highly related receptor tyrosine kinases, TIE and TEK, comprise a family of endothelial cell-specific kinase. We established monoclonal antibodies against them and performed detailed analyses on their expression and function in murine hematopoietic stem cells (HSCs). TIE and TEK were expressed on 23.7% and 33.3% of lineage marker-negative, c-Kit+ and Sca-1+ (Lin− c-Kit+ Sca-1+) HSCs that contain the majority of day-12 colony-forming units-spleen (CFU-S) and long-term reconstituting cells, but not committed progenitor cells. Lin− c-Kit+ Sca-1+ cells were further divided by the expression of TIE and TEK. TIE+ and TEK+ HSCs as well as each negative counterpart contained high proliferative potential colony-forming cells and differentiated into lymphoid and myeloid progenies both in vitro and in vivo. However, day-12 CFU-S were enriched in TIE+ and TEK+ HSCs. Our findings define TIE and TEK as novel stem cell marker antigens that segregate day-12 CFU-S, and provide evidence of novel signaling pathways that are involved in the functional regulation of HSCs at a specific stage of differentiation, particularly of day-12 CFU-S.

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